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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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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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.
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 Figs. 1-25, Tables 1–3 and methods.
Sequences of carboxylic acid reductases from homology search for SSN calculation.
Docking model of CoA-SH into CARsr-A.
Docking model of pantetheine into CARsr-A.
Docking model of SNAC into CARsr-A.
Amino acid sequences of enzymes used for this study.
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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