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Engineered the Active Site of ω-Transaminase for Enhanced Asymmetric Synthesis Towards (S)-1-[4-(Trifluoromethyl)phenyl]ethylamine

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Abstract

ω-Transaminase (ω-TA) is a promising biocatalyst for the synthesis of chiral amines. In this study, a ω-TA derived from Vitreoscilla stercoraria DSM 513 (VsTA) was heterologous expressed in recombinant E. coli cells and applied to reduce 4′-(trifluoromethyl)acetophenone (TAP) to (S)-1-[4-(trifluoromethyl)phenyl]ethylamine ((S)-TPE), a pharmaceutical intermediate of chiral amine. Aimed to a more efficient synthesis of (S)-TPE, VsTA was further engineered via a semi-rational strategy. Compared to wild-type VsTA, the obtained R411A variant exhibited 2.39 times higher activity towards TAP and enhanced catalytic activities towards other prochiral aromatic ketones. Additionally, better thermal stability for R411A variant was observed with 25.4% and 16.3% increase in half-life at 30 °C and 40 °C, respectively. Structure-guided analysis revealed that the activity improvement of R411A variant was attributed to the introduction of residue A411, which is responsible for the increase in the hydrophobicity of substrate tunnel and the alleviation of steric hindrance, thereby facilitating the accessibility of hydrophobic substrate TAP to the active center of VsTA. This study provides an efficient strategy for the engineering of ω-TA based on semi-rational approach and has the potential for the molecular modification of other biocatalysts.

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All data generated or analyzed during this study are included in this published article and its supplementary information file.

References

  1. Kelly, S. A., Pohle, S., Wharry, S., Mix, S., Allen, C. C. R., Moody, T. S., & Gilmore, B. F. (2018). Application of ω-transaminases in the pharmaceutical industry. Chemical Reviews, 118, 349–367. https://doi.org/10.1021/acs.chemrev.7b00437

    Article  CAS  PubMed  Google Scholar 

  2. Savile, C. K., Janey, J. M., Mundorff, E. C., Moore, J. C., Tam, S., Jarvis, W. R., Colbeck, J. C., Krebber, A., Fleitz, F. J., Brands, J., Devine, P. N., Huisman, G. W., & Hughes, G. J. (2010). Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science, 329, 305–309. https://doi.org/10.1126/science.1188934

    Article  ADS  CAS  PubMed  Google Scholar 

  3. Novick, S. J., Dellas, N., Garcia, R., Ching, C., Bautista, A., Homan, D., Alvizo, O., Entwistle, D., Kleinbeck, F., Schlama, T., & Ruch, T. (2021). Engineering an amine transaminase for the efficient production of a chiral sacubitril precursor. ACS Catalysis, 11, 3762–3770. https://doi.org/10.1021/acscatal.0c05450

    Article  CAS  Google Scholar 

  4. Schober, M., MacDermaid, C., Ollis, A. A., Chang, S., Khan, D., Hosford, J., Latham, J., Ihnken, L. A. F., Brown, M. J. B., Fuerst, D., Sanganee, M. J., & Roiban, G. D. (2019). Chiral synthesis of LSD1 inhibitor GSK2879552 enabled by directed evolution of an imine reductase. Nature Catalysis, 2, 909–915. https://doi.org/10.1038/s41929-019-0341-4

    Article  CAS  Google Scholar 

  5. Cabré, A., Verdaguer, X., & Riera, A. (2022). Recent advances in the enantioselective synthesis of chiral amines via transition metal-catalyzed asymmetric hydrogenation. Chemical Reviews, 122, 269–339. https://doi.org/10.1021/acs.chemrev.1c00496

    Article  CAS  PubMed  Google Scholar 

  6. Kohls, H., Steffen-Munsberg, F., & Höhne, M. (2014). Recent achievements in developing the biocatalytic toolbox for chiral amine synthesis. Current Opinion in Chemical Biology, 19, 180–192. https://doi.org/10.1016/j.cbpa.2014.02.021

    Article  CAS  PubMed  Google Scholar 

  7. Patil, M. D., Grogan, G., Bommarius, A., & Yun, H. (2018). Oxidoreductase-catalyzed synthesis of chiral amines. ACS Catalysis, 8, 10985–11015. https://doi.org/10.1021/acscatal.8b02924

    Article  CAS  Google Scholar 

  8. Fuchs, M., Farnberger, J. E., & Kroutil, W. (2015). The industrial age of biocatalytic transamination. European Journal of Organic Chemistry, 2015, 6965–6982. https://doi.org/10.1002/ejoc.201500852

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ferrandi, E. E., & Monti, D. (2018). Amine transaminases in chiral amines synthesis: Recent advances and challenges. World Journal of Microbiology and Biotechnology, 34, 13. https://doi.org/10.1007/s11274-017-2395-2

    Article  CAS  Google Scholar 

  10. Gomm, A., & O’Reilly, E. (2018). Transaminases for chiral amine synthesis. Current Opinion in Chemical Biology, 43, 106–112. https://doi.org/10.1016/j.cbpa.2017.12.007

    Article  CAS  PubMed  Google Scholar 

  11. Ghislieri, D., & Turner, N. J. (2014). Biocatalytic approaches to the synthesis of enantiomerically pure chiral amines. Topics in Catalysis, 57, 284–300. https://doi.org/10.1007/s11244-013-0184-1

    Article  CAS  Google Scholar 

  12. Guo, F., & Berglund, P. (2017). Transaminase biocatalysis: Optimization and application. Green Chemistry, 19, 333–360. https://doi.org/10.1039/c6gc02328b

    Article  CAS  Google Scholar 

  13. Li, F., Du, Y., Liang, Y., Wei, Y., Zheng, Y., & Yu, H. (2023). Redesigning an (R)-selective transaminase for the efficient synthesis of pharmaceutical N-heterocyclic amines. ACS Catalysis, 13, 422–432. https://doi.org/10.1021/acscatal.2c05177

    Article  CAS  Google Scholar 

  14. Slabu, I., Galman, J. L., Lloyd, R. C., & Turner, N. J. (2017). Discovery, engineering, and synthetic application of transaminase biocatalysts. ACS Catalysis, 7, 8263–8284. https://doi.org/10.1021/acscatal.7b02686

    Article  CAS  Google Scholar 

  15. Qu, G., Li, A., Acevedo-Rocha, C. G., Sun, Z., & Reetz, M. T. (2020). The crucial role of methodology development in directed evolution of selective enzymes. Angewandte Chemie- International Edition, 59, 13204–13231. https://doi.org/10.1002/anie.201901491

    Article  CAS  PubMed  Google Scholar 

  16. Morley, K. L., & Kazlauskas, R. J. (2005). Improving enzyme properties: When are closer mutations better? Trends in Biotechnology, 23, 231–237. https://doi.org/10.1016/j.tibtech.2005.03.005

    Article  CAS  PubMed  Google Scholar 

  17. Shin, J. S., & Kim, B. G. (2002). Exploring the active site of amine:Pyruvate aminotransferase on the basis of the substrate structure-reactivity relationship: How the enzyme controls substrate specificity and stereoselectivity. Journal of Organic Chemistry, 67, 2848–2853. https://doi.org/10.1021/jo016115i

    Article  CAS  PubMed  Google Scholar 

  18. Meng, Q., Ramírez-Palacios, C., Capra, N., Hooghwinkel, M. E., Thallmair, S., Rozeboom, H. J., Thunnissen, A. M. W. H., Wijma, H. J., Marrink, S. J., & Janssen, D. B. (2021). Computational redesign of an ω-transaminase from Pseudomonas jessenii for asymmetric synthesis of enantiopure bulky amines. ACS Catalysis, 11, 10733–10747. https://doi.org/10.1021/acscatal.1c02053

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nobili, A., Steffen-Munsberg, F., Kohls, H., Trentin, I., Schulzke, C., Höhne, M., & Bornscheuer, U. T. (2015). Engineering the active site of the amine transaminase from Vibrio fluvialis for the asymmetric synthesis of aryl-alkyl amines and amino alcohols. ChemCatChem, 7, 757–760. https://doi.org/10.1002/cctc.201403010

    Article  CAS  Google Scholar 

  20. Land, H., Ruggieri, F., Szekrenyi, A., Fessner, W. D., & Berglund, P. (2020). Engineering the active site of an (S)-selective amine transaminase for acceptance of doubly bulky primary amines. Advanced Synthesis and Catalysis, 362, 812–821. https://doi.org/10.1002/adsc.201901252

    Article  CAS  Google Scholar 

  21. Xie, Y., Xu, F., Yang, L., Liu, H., Xu, X., Wang, H., & Wei, D. (2021). Engineering the large pocket of an (S)-selective transaminase for asymmetric synthesis of (S)-1-amino-1-phenylpropane. Catalysis Science and Technology, 11, 2461–2470. https://doi.org/10.1039/d0cy02426k

    Article  ADS  CAS  Google Scholar 

  22. Ma, Y., Jiao, X., Wang, Z., Mu, H., Sun, K., Li, X., Zhao, T., Liu, X., & Zhang, N. (2022). Engineering a transaminase for the efficient synthesis of a key intermediate for rimegepant. Organic Process Research and Development, 26, 1971–1977. https://doi.org/10.1021/acs.oprd.1c00376

    Article  CAS  Google Scholar 

  23. Jia, D. X., Peng, C., Li, J. L., Wang, F., Liu, Z. Q., & Zheng, Y. G. (2021). Redesign of (R)-omega-transaminase and its application for synthesizing amino acids with bulky side chain. Applied Biochemistry and Biotechnology, 193, 3624–3640. https://doi.org/10.1007/s12010-021-03616-7

    Article  CAS  PubMed  Google Scholar 

  24. Wang, Y., Feng, J., Dong, W., Chen, X., Yao, P., Wu, Q., & Zhu, D. (2021). Improving catalytic activity and reversing enantio-specificity of ω-transaminase by semi-rational engineering en route to chiral bulky β-amino esters. ChemCatChem, 13, 3396–3400. https://doi.org/10.1002/cctc.202100503

    Article  CAS  Google Scholar 

  25. Han, R., Cao, X., Fang, H., Zhou, J., & Ni, Y. (2021). Structure-based engineering of ω-transaminase for enhanced catalytic efficiency toward (R)-(+)-1-(1-naphthyl)ethylamine synthesis. Molecular Catalysis, 502, 111368. https://doi.org/10.1016/j.mcat.2020.111368

    Article  CAS  Google Scholar 

  26. Cheng, F., Chen, X. L., Li, M. Y., Zhang, X. J., Jia, D. X., Wang, Y. J., Liu, Z. Q., & Zheng, Y. G. (2020). Creation of a robust and R-selective ω-amine transaminase for the asymmetric synthesis of sitagliptin intermediate on a kilogram scale. Enzyme and Microbial Technology, 141, 109655. https://doi.org/10.1016/j.enzmictec.2020.109655

    Article  CAS  PubMed  Google Scholar 

  27. Yang, X., Wu, T., Phipps, R. J., & Toste, F. D. (2015). Advances in catalytic enantioselective fluorination, mono-, di-, and trifluoromethylation, and trifluoromethylthiolation reactions. Chemical Reviews, 115, 826–870. https://doi.org/10.1021/cr500277b

    Article  CAS  PubMed  Google Scholar 

  28. Zhou, Y., Wang, J., Gu, Z., Wang, S., Zhu, W., Acenã, J. L., Soloshonok, V. A., Izawa, K., & Liu, H. (2016). Next generation of fluorine-containing pharmaceuticals, compounds currently in phase II-III clinical trials of major pharmaceutical companies: New structural trends and therapeutic areas. Chemical Reviews, 116, 422–518. https://doi.org/10.1021/acs.chemrev.5b00392

    Article  CAS  PubMed  Google Scholar 

  29. Cardiac sarcomere inhibitors. (2022). US Patent No 11414424 B2. Available from: https://ppubs.uspto.gov/dirsearch-public/print/downloadPdf/11414424. Accessed 18 Feb 2024

  30. Indole carboxamide derivative and pharmaceutical composition containing same. (2023). US Patent No 20230000821 A1. Available from: https://ppubs.uspto.gov/dirsearch-public/print/downloadPdf/20230000821. Accessed 18 Feb 2024

  31. Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F. T., Beer, T. A. P., Rempfer, C., Bordoli, L., Lepore, R., & Schwede, T. (2018). SWISS-MODEL: Homology modelling of protein structures and complexes. Nucleic Acids Research, 46, W296–W303. https://doi.org/10.1093/nar/gky427

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Laskowski, R. A., MacArthur, M. W., Moss, D. S., & Thornton, J. M. (1993). PROCHECK: A program to check the stereochemical quality of protein structures. Journal of Applied Crystallography, 26, 283–291. https://doi.org/10.1107/s0021889892009944

    Article  ADS  CAS  Google Scholar 

  33. Colovos, C., & Yeates, T. O. (1993). Verification of protein structures: Patterns of nonbonded atomic interactions. Protein Science, 2, 1511–1519. https://doi.org/10.1002/pro.5560020916

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Forli, S., Huey, R., Pique, M. E., Sanner, M. F., Goodsell, D. S., & Olson, A. J. (2016). Computational protein-ligand docking and virtual drug screening with the AutoDock suite. Nature Protocols, 11, 905–919. https://doi.org/10.1038/nprot.2016.051

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Morrison, K. L., & Weiss, G. A. (2001). Combinatorial alanine-scanning. Current Opinion in Chemical Biology, 5, 302–307. https://doi.org/10.1016/S1367-5931(00)00206-4

    Article  CAS  PubMed  Google Scholar 

  36. Chovancova, E., Pavelka, A., Benes, P., Strnad, O., Brezovsky, J., Kozlikova, B., Gora, A., Sustr, V., Klvana, M., Medek, P., Biedermannova, L., Sochor, J., & Damborsky, J. (2012). CAVER 30: A tool for the analysis of transport pathways in dynamic protein structures. PLoS Computational Biology, 8, e1002708. https://doi.org/10.1371/journal.pcbi.1002708

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Xiang, C., Ao, Y. F., Höhne, M., & Bornscheuer, U. T. (2022). Shifting the pH optima of (R)-selective transaminases by protein engineering. International Journal of Molecular Sciences, 23, 15347. https://doi.org/10.3390/ijms232315347

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kelefiotis-Stratidakis, P., Tyrikos-Ergas, T., & Pavlidis, I. V. (2019). The challenge of using isopropylamine as an amine donor in transaminase catalysed reactions. Organic and Biomolecular Chemistry, 17, 1634–1642. https://doi.org/10.1039/c8ob02342e

    Article  CAS  PubMed  Google Scholar 

  39. Contente, M. L., Planchestainer, M., Molinari, F., & Paradisi, F. (2016). Stereoelectronic effects in the reaction of aromatic substrates catalysed by: Halomonas elongata transaminase and its mutants. Organic and Biomolecular Chemistry, 14, 9306–9311. https://doi.org/10.1039/c6ob01629d

    Article  CAS  PubMed  Google Scholar 

  40. Rodrigues, C. J. C., Ferrer, M., & de Carvalho, C. C. C. R. (2021). ω-Transaminase-mediated asymmetric synthesis of (S)-1-(4-trifluoromethylphenyl)ethylamine. Catalysts, 11, 307. https://doi.org/10.3390/catal11030307

    Article  CAS  Google Scholar 

  41. López-Iglesias, M., González-Martínez, D., Rodríguez-Mata, M., Gotor, V., Busto, E., Kroutil, W., & Gotor-Fernández, V. (2017). Asymmetric biocatalytic synthesis of fluorinated pyridines through transesterification or transamination: Computational insights into the reactivity of transaminases. Advanced Synthesis & Catalysis, 359, 279–291. https://doi.org/10.1002/adsc.201600835

    Article  CAS  Google Scholar 

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Funding

This work was financially supported by grants from the National Key Research and Development Program of China (2022YFA0911804), the National Natural Science Foundation of China (No. 21676250), and the Zhejiang Province Natural Science Foundation of China (LY22B060010).

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Authors and Affiliations

  1. Key Laboratory for Green Pharmaceutical Technologies and Related Equipment of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Zhi-Wen Duan, Yao-Wu Wang & Pu Wang

  2. Key Laboratory of Pharmaceutical Engineering of Zhejiang Province, College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Zhi-Wen Duan, Yao-Wu Wang & Pu Wang

  3. Research & Development Center, Zhejiang Medicine Co. Ltd., Shaoxing, 312500, People’s Republic of China

    Da-Dong Shen & Xin-Qiang Sun

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Contributions

ZD: visualization, writing—original draft, data curation, conceptualization, writing—review and editing. YW: data curation, investigation. DS: data curation. XS: writing—review and editing. PW: conceptualization, resources, project administration, funding acquisition, supervision.

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Correspondence to Pu Wang.

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Duan, ZW., Wang, YW., Shen, DD. et al. Engineered the Active Site of ω-Transaminase for Enhanced Asymmetric Synthesis Towards (S)-1-[4-(Trifluoromethyl)phenyl]ethylamine. Appl Biochem Biotechnol (2024). https://doi.org/10.1007/s12010-024-04886-7

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