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Biocatalytic synthesis of non-standard amino acids by a decarboxylative aldol reaction

Abstract

Enzymes are renowned for their catalytic efficiency and selectivity. Despite the wealth of carbon–carbon bond-forming transformations in traditional organic chemistry and nature, relatively few C–C bond-forming enzymes have found their way into the biocatalysis toolbox. Here we show that the enzyme UstD performs a highly selective decarboxylative aldol addition with diverse aldehyde substrates to make non-standard γ-hydroxy amino acids. We increased the activity of UstD through three rounds of classic directed evolution and an additional round of computationally guided engineering. The enzyme that emerged, UstDv2.0, is efficient in a whole-cell biocatalysis format. The products are highly desirable, functionally rich bioactive γ-hydroxy amino acids that we demonstrate can be prepared stereoselectively on the gram scale. The X-ray crystal structure of UstDv2.0 at 2.25 Å reveals the active site and provides a foundation for probing the UstD mechanism.

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Fig. 1: Relevance and mechanism of enzymatic C–C bond formation.
Fig. 2: Directed evolution of UstD and the evaluation of variants.
Fig. 3: Engineering UstD for an increased crystallizability and activity in whole-cell catalysis.

Data availability

The structure of UstDv2.0 is available through the Protein Data Bank ID 7MKV. The sequence-activity data used for linear regression modelling is available through GitHub42. All the other data are available from the authors upon reasonable request.

Code availability

The linear regression modelling code used during the final round of protein engineering is available through GitHub42 under the MIT License.

References

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

    CAS  Google Scholar 

  2. Zhang, X. et al. Divergent synthesis of complex diterpenes through a hybrid oxidative approach. Science 369, 799–806 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Brown, D. G. & Boström, J. Analysis of past and present synthetic methodologies on medicinal chemistry: where have all the new reactions gone? J. Med. Chem. 59, 4443–4458 (2016).

    CAS  PubMed  Google Scholar 

  4. Fesko, K. & Gruber-Khadjawi, M. Biocatalytic methods for C–C bond formation. ChemCatChem 5, 1248–1272 (2013).

    CAS  Google Scholar 

  5. Schmidt, N. G., Eger, E. & Kroutil, W. Building bridges: biocatalytic C–C-bond formation toward multifunctional products. ACS Catal. 6, 4286–4311 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Fujii, I. Heterologous expression systems for polyketide synthases. Nat. Prod. Rep. 26, 155–169 (2009).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  8. Wang, Z. J. et al. Improved cyclopropanation activity of histidine-ligated cytochrome P450 enables the enantioselective formal synthesis of levomilnacipran. Angew. Chem. Int. Ed. 53, 6810–6813 (2014).

    CAS  Google Scholar 

  9. Berkeš, D., Kolarovič, A., Manduch, R., Baran, P. & Považanec, F. Crystallization-induced asymmetric transformations (CIAT): stereoconvergent acid-catalyzed lactonization of substituted 2-amino-4-aryl-4-hydroxybutanoic acids. Tetrahedron Asymm. 16, 1927–1934 (2005).

    Google Scholar 

  10. Goldberg, S. L. et al. Preparation of β-hydroxy-α-amino acid using recombinant d-threonine aldolase. Org. Process Res. Dev. 19, 1308–1316 (2015).

    CAS  Google Scholar 

  11. Steinreiber, J. et al. Overcoming thermodynamic and kinetic limitations of aldolase-catalyzed reactions by applying multienzymatic dynamic kinetic asymmetric transformations. Angew. Chem. Int. Ed. 46, 1624–1626 (2007).

    CAS  Google Scholar 

  12. Zetzsche, L. E. & Narayan, A. R. H. Broadening the scope of biocatalytic C–C bond formation. Nat. Rev. Chem. 4, 334–346 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Ye, Y. et al. Unveiling the biosynthetic pathway of the ribosomally synthesized and post-translationally modified peptide ustiloxin B in filamentous fungi. Angew. Chem. Int. Ed. 55, 8072–8075 (2016).

    CAS  Google Scholar 

  14. Prier, C. K. & Arnold, F. H. Chemomimetic biocatalysis: exploiting the synthetic potential of cofactor-dependent enzymes to create new catalysts. J. Am. Chem. Soc. 137, 13992–14006 (2015).

    CAS  PubMed  Google Scholar 

  15. Di Salvo, M. L. et al. On the catalytic mechanism and stereospecificity of Escherichia coli l-threonine aldolase. FEBS J. 281, 129–145 (2014).

    PubMed  Google Scholar 

  16. Marsden, S. R., Gjonaj, L., Eustace, S. J. & Hanefeld, U. Separating thermodynamics from kinetics—a new understanding of the transketolase reaction. ChemCatChem 9, 1808–1814 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Ariza, J., Font, J. & Ortuño, R. M. An efficient and concise entry to (–)-4,5-dihydroxy-d-threo-l-norvaline. Formal synthesis of clavalanine. Tetrahedron Lett. 32, 1979–1982 (1991).

    CAS  Google Scholar 

  18. Blaskovich, M. A. T. Unusual amino acids in medicinal chemistry. J. Med. Chem. 59, 10807–10836 (2016).

    CAS  PubMed  Google Scholar 

  19. Moreno, C. J. et al. Synthesis of γ-hydroxy-α-amino acid derivatives by enzymatic tandem aldol addition–transamination reactions. ACS Catal. 11, 4660–4669 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Hernandez, K. et al. Combining aldolases and transaminases for the synthesis of 2-amino-4-hydroxybutanoic acid. ACS Catal. 7, 1707–1711 (2017).

    CAS  Google Scholar 

  21. Vargas-Rodriguez, O., Sevostyanova, A., Söll, D. & Crnković, A. Upgrading aminoacyl-tRNA synthetases for genetic code expansion. Curr. Opin. Chem. Biol. 46, 115–122 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Marchand, J. A. et al. Discovery of a pathway for terminal-alkyne amino acid biosynthesis. Nature 567, 420–424 (2019).

    CAS  PubMed  Google Scholar 

  23. Yang, J. et al. The I-TASSER suite: protein structure and function prediction. Nat. Methods 12, 7–8 (2014).

    Google Scholar 

  24. Ho, T. H. et al. Catalytic intermediate crystal structures of cysteine desulfurase from the Archaeon Thermococcus onnurineus NA1. Archaea 2017, 1–11 (2017).

    CAS  Google Scholar 

  25. Kumar, P. et al. l-Threonine transaldolase activity is enabled by a persistent catalytic intermediate. ACS Chem. Biol. 16, 95 (2021).

    Google Scholar 

  26. Reetz, M. T., Prasad, S., Carballeira, J. D., Gumulya, Y. & Bocola, M. Iterative saturation mutagenesis accelerates laboratory evolution of enzyme stereoselectivity: rigorous comparison with traditional methods. J. Am. Chem. Soc. 132, 9144–9152 (2010).

    CAS  PubMed  Google Scholar 

  27. Romero, P. A. & Arnold, F. H. Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10, 866–876 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 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).

    CAS  Google Scholar 

  29. Romney, D. K., Sarai, N. S. & Arnold, F. H. Nitroalkanes as versatile nucleophiles for enzymatic synthesis of noncanonical amino acids. ACS Catal. 9, 8726–8730 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Marfey, P. Determination of d-amino acids. II. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res. Commun. 49, 591–596 (1984).

    CAS  Google Scholar 

  31. Wu, G. et al. Proline and hydroxyproline metabolism: implications for animal and human nutrition. Amino Acids 40, 1053–1063 (2011).

    CAS  PubMed  Google Scholar 

  32. Müller, J.-C., Toome, V., Pruess, D. L., Blount, J. F. & Weigele, M. Ro 22-5417, a new clavam antibiotic from Streptomyces clavuligerus. III Absolute stereochemistry. J. Antibiot. 36, 217–225 (1983).

    Google Scholar 

  33. Wahab, R. A., Elias, N., Abdullah, F. & Ghoshal, S. K. On the taught new tricks of enzymes immobilization: an all-inclusive overview. React. Func. Pol. 152, 104613 (2020).

    CAS  Google Scholar 

  34. Wachtmeister, J. & Rother, D. Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale. Curr. Opin. Biotechnol. 42, 169–177 (2016).

    CAS  PubMed  Google Scholar 

  35. Al-Ayyoubi, M., Gettins, P. G. W. & Volz, K. Crystal structure of human maspin, a serpin with antitumor properties: reactive center loop of maspin is exposed but constrained. J. Biol. Chem. 279, 55540–55544 (2004).

    CAS  PubMed  Google Scholar 

  36. Fox, R. Directed molecular evolution by machine learning and the influence of nonlinear interactions. J. Theor. Biol. 234, 187–199 (2005).

    CAS  PubMed  Google Scholar 

  37. Huffman, M. A. et al. Design of an in vitro biocatalytic cascade for the manufacture of islatravir. Science 366, 1255–1259 (2019).

    CAS  PubMed  Google Scholar 

  38. Eliot, A. C. & Kirsch, J. F. Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Annu. Rev. Biochem. 73, 383–415 (2004).

    CAS  PubMed  Google Scholar 

  39. Romney, D. K., Murciano-Calles, J., Wehrmüller, J. E. & Arnold, F. H. Unlocking reactivity of TrpB: a general biocatalytic platform for synthesis of tryptophan analogues. J. Am. Chem. Soc. 139, 10769–10776 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Boville, C. E. et al. Engineered biosynthesis of β-alkyl tryptophan analogues. Angew. Chem. Int. Ed. 57, 14764–14768 (2018).

    CAS  Google Scholar 

  41. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    CAS  PubMed  Google Scholar 

  42. Ellis, J. M. Linear regression analysis of UstD-TLM. Zenodo https://doi.org/10.5281/zenodo.5719389 (2021).

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Acknowledgements

We thank I. Guzei for small-molecule X-ray structure determinations and S.H. Gellman and members of the Buller group for critical reading of the manuscript. The crystal mounting and data collection were mediated by the Collaborative Crystallography Core, Department of Biochemistry, UW–Madison, and data were collected at the Life Sciences Collaborative Access Team beamline 21ID-D at the Advanced Photon Source, Argonne National Laboratory, and we thank Z. Wawrzak for technical assistance during data collection. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). This work was supported by the Office of the Vice Chancellor for Research and Graduate Education at the University of Wisconsin-Madison, Wisconsin Alumni Research Foundation, National Institute of Health (grant DP2-GM137417, A.R.B.), Morgridge Institute for Research—Metabolism Theme Fellowship (P.K.) and the NIH Biotechnology Training Grant (T32-GM008349, J.M.E.). The Bruker AVANCE III-500 NMR spectrometers were supported by the Bender Fund. The Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38. The Bruker D8 VENTURE Photon III X-ray diffractometer was partially funded by a NSF Award (no. CHE-1919350) to the UW–Madison Department of Chemistry.

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Contributions

A.R.B. and J.M.E. conceptualized the goals and aims of the project. J.M.E., M.E.C., P.K., E.P.G., C.A.B. and A.R.B. carried out the development of the chemistry and enzymes. J.M.E. developed the code for data analysis and developed the linear regression model. J.M.E. and M.E.C. verified the results. J.M.E., M.E.C., P.K. and A.R.B. prepared the figures and data visualizations. A.R.B. secured funding for the project that led to this publication. A.R.B. coordinated team members for the development of the chemistry and enzyme evolution. C.A.B. supervised the data acquisition of protein crystals that led to the resolved crystal structure. A.R.B. supervised the research activity planning and execution. J.M.E., M.E.C. and A.R.B. prepared the initial manuscript. J.M.E., M.E.C., P.K. and A.R.B. reviewed and edited the initial manuscript and provided critical commentary and revisions.

Corresponding author

Correspondence to Andrew R. Buller.

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Competing interests

A.R.B., J.M.E. and P.K. have a patent pending on the use of engineered UstD for the synthesis of nsAAs, US Patent application no. 20210115480A1. All other authors declare no competing interests.

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Ellis, J.M., Campbell, M.E., Kumar, P. et al. Biocatalytic synthesis of non-standard amino acids by a decarboxylative aldol reaction. Nat Catal 5, 136–143 (2022). https://doi.org/10.1038/s41929-022-00743-0

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