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Thioester-mediated biocatalytic amide bond synthesis with in situ thiol recycling

Nature Catalysis volume 6, pages 89–99 (2023)Cite this article

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Abstract

The activation of carboxylic acids to thioesters plays an important role in biology. However, biochemical studies and biotechnological applications are hampered by a general lack of access to thioesters, especially those based on Coenzyme A (CoA-SH). Here we show a generic thioester recycling enzyme by exploiting the promiscuous activity of a carboxylic acid reductase (CARsr). The adenylation domain of CARsr (CARsr-A) catalyses the conversion of a wide range of carboxylic acids to acyl-S-Coenzyme A and other thioesters in good yields. CARsr-A was used in situ as part of a recycling system to regenerate thioesters for acyl-S-Coenzyme A-dependent enzymes in one-pot reactions. This concept of thioester recycling is demonstrated with a range of acyltransferases that allow the formation of diverse amides and the non-native acylation of lysine side chains in a histone-derived peptide using the epigenetic writer, lysine acetyltransferase HATp300. Overall, these results establish a generic platform for thioester formation towards amide formation and beyond.

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Fig. 1: Generation and recycling of acyl-S-CoA thioesters using a carboxylic acid reductase A-domain in amide bond formations.
Fig. 2: Identification of CoA-SH binding in CARsr-A and the enzymatic formation of different thioesters.
Fig. 3: A broad range of acyl-S-CoA thioesters is formed by CARsr-A.
Fig. 4: Amide bond formation by combining acyl-S-CoA dependent ATs with thioester regeneration system.
Fig. 5: Acylation of histone peptide H4 (1–20) via the formation of non-native acyl-S-CoA esters.

Data availability

The data supporting the findings of this study are available within the article, its Supplementary Information, on https://doi.org/10.6084/m9.figshare.21277920.v2 or can be obtained from the corresponding author upon reasonable request.

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Acknowledgements

We acknowledge funding by the EPSRC, BBSRC and AstraZeneca plc under the Prosperity Partnership EP/S005226/1. M.L. thanks CoEBio3 for funding for a studentship. We are grateful to W. Goundry for helpful discussions and thank R. Sung and R. Spiess for support with the MS analyses.

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Authors and Affiliations

  1. Department of Chemistry, The University of Manchester, Manchester Institute of Biotechnology, Manchester, UK

    Christian Schnepel, Laura Rodríguez Pérez, Yuqi Yu, Antonio Angelastro, Rachel S. Heath, Max Lubberink, Nicholas J. Turner & Sabine L. Flitsch

  2. Early Chemical Development, Pharmaceutical Sciences, R&D, AstraZeneca, Cambridge, UK

    Francesco Falcioni

  3. Chemical Development, Pharmaceutical Technology & Development, Operations, AstraZeneca, Macclesfield, UK

    Keith Mulholland

  4. Compound Synthesis and Management, Discovery Sciences, R&D, AstraZeneca, Gothenburg, Sweden

    Martin A. Hayes

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Contributions

S.L.F., N.J.T, M.A.H., K.M. and F.F. managed and supervised the project. S.L.F, N.J.T., C.S. and M.A.H. devised the concept. C.S. performed the cloning, biocatalyst production, enzyme assays and thioester scope studies. C.S. undertook and optimized the cascade reactions and screenings. L.R.P. and A.A. carried out the histone acylation assays. Y.Y. performed the bioinformatic studies. C.S. synthesized the amide products, and C.S. and A.A. performed the purification of the products. C.S. and M.L. designed the A-domain and did the initial activity tests. C.S. and R.S.H. carried out the enzyme purifications. C.S., S.L.F., L.R.P., N.J.T. and Y.Y. wrote the manuscript and generated the figures.

Corresponding authors

Correspondence to Christian Schnepel or Sabine L. Flitsch.

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The authors declare no competing interests.

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Nature Catalysis thanks Francesca Paradisi, Wei Niu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1-25, Tables 1–3 and methods.

Reporting Summary

Supplementary Data 1

Sequences of carboxylic acid reductases from homology search for SSN calculation.

Supplementary Data 2

Docking model of CoA-SH into CARsr-A.

Supplementary Data 3

Docking model of pantetheine into CARsr-A.

Supplementary Data 4

Docking model of SNAC into CARsr-A.

Supplementary Data 5

Amino acid sequences of enzymes used for this study.

Supplementary Data 6

Vector construct encoding A-domain of carboxylic acid reductase from Segniliparus rugosus.

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Schnepel, C., Pérez, L.R., Yu, Y. et al. Thioester-mediated biocatalytic amide bond synthesis with in situ thiol recycling. Nat Catal 6, 89–99 (2023). https://doi.org/10.1038/s41929-022-00889-x

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