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A strategic Ser/Cys exchange in the catalytic triad unlocks an acyltransferase-mediated synthesis of thioesters and tertiary amides

Abstract

Expanding the toolbox of enzymatic reactions accessible to organic chemists is one of the major goals in biocatalysis. Here we describe the development of an acyltransferase variant from Mycobacterium smegmatis in which a strategic Ser/Cys exchange in the catalytic triad dramatically expanded its synthetic capability to yield a biocatalyst able to efficiently catalyse the formation of thioesters and tertiary amides in water. Preparative scale (250 mM) biotransformations were performed starting from different thiols and secondary amines with excellent yields and reactions times, using vinyl esters as acylating agents. The high substrate-to-catalyst ratio and the cofactor independence make this process a sustainable and cost-effective procedure that was successfully applied to the synthesis of acetyl coenzyme A as well as structurally simpler analogues. Computational studies provided insights into the enzymatic selectivity and substrate recognition.

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Fig. 1: Biosynthesis of acetyl-CoA and its mimetics.
Fig. 2: Docking of acetyl-CoA.
Fig. 3: CoA and acetyl-CoA computational studies.

Data availability

All data are available from the corresponding authors upon request. The crystal structure used as the starting point for the in silico analysis is available in the Protein Data Bank (https://www.rcsb.org/) under accession no. 2Q0S. The dataset for conservation analysis can be found in https://pfam.xfam.org/family/PF13472.

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Acknowledgements

This project was supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 792804 AROMAs-FLOW (M.L.C.), and SNSF (200021_192274, F.P.). The authors thank P. Renaud (DCB, University of Bern) for insightful scientific discussions.

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Authors and Affiliations

Authors

Contributions

F.P. developed the original concept. M.L.C. performed the experimental work and analysed the results. D.R.-P. conceived and performed the computational experiments. M.L.C., F.M. and F.P. conceived and designed the experiments. All the authors co-wrote the manuscript.

Corresponding authors

Correspondence to Francesco Molinari or Francesca Paradisi.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Catalysis thanks Luis Morales-Quintana, Nicholas Turner 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 Methods, Figs. 1–9 and references.

Reporting Summary

Supplementary Video 1

Depiction of the transport of CoA from the bulk to the active site and acetyl CoA back outside in the tunnel identified.

Supplementary Data 1

Initial and final coordinates of the systems in the molecular dynamics simulations as pdb formatted files.

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Contente, M.L., Roura Padrosa, D., Molinari, F. et al. A strategic Ser/Cys exchange in the catalytic triad unlocks an acyltransferase-mediated synthesis of thioesters and tertiary amides. Nat Catal 3, 1020–1026 (2020). https://doi.org/10.1038/s41929-020-00539-0

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