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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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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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.
The authors declare no competing interests.
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 Methods, Figs. 1–9 and references.
Depiction of the transport of CoA from the bulk to the active site and acetyl CoA back outside in the tunnel identified.
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