Abstract
Reductive desymmetrization of 2,2-disubstituted prochiral 1,3-cyclodiketones to 2,2-disubstituted-3-hydroxycycloketones is a highly desired transformation for the construction of complex molecules with multiple chiral centres, but the generation of a single stereoisomer is difficult and an extremely challenging task in organic chemistry. In this study, by using ethyl secodione as the model substrate and an engineered carbonyl reductase from Ralstonia sp. as the biocatalyst, we realized the efficient reductive desymmetrization of 2,2-disubstituted cyclodiketones to give essentially one single stereoisomer. The mutant enzyme F12 (I91V/I187S/I188L/Q191N/F205A) showed an 183-fold enhancement of enzyme activity and outstanding stereoselectivity towards most of the tested prochiral 1,3-cyclodiketones. Crystal structural analysis and molecular dynamics studies reveal the molecular basis for activity improvement and the stereoselectivity control mechanism. Our results show that by altering the active site conformation populations (particularly the position of an α-helix) to properly accommodate the larger substrate and co-factor for catalysis, this challenging synthetic problem can ultimately be addressed.
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Acknowledgements
We would like to thank L. Pu of the Department of Chemistry, University of Virginia for the helpful discussions about the chirality of the products. This work was financially supported by the National Key R&D Program of China (no. 2018YFA0901600), National Natural Science Foundation of China (no. 21602246) and Tianjin Municipal Science and Technology Commission (nos 15PTGCCX00060 and 15PTCYSY00020). M.A.M.S. is grateful to the Spanish MINECO for a PhD fellowship (BES-2015-074964). S.O. thanks the funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC-2015-StG-679001) and the Spanish MINECO for I+D project PGC2018-102192-B-I00. M.A.M.S. and S.O. thank the Generalitat de Catalunya for the group-emergent CompBioLab (2017 SGR-1707).
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Contributions
X.C., H.Z., Q.W., Y.M. and D.Z. conceived and designed the project. X.C. synthesized the compounds, purified the products and analysed the chirality of all of the products. H.Z. designed and performed the mutation, screening and growing crystal experiments. M.A.M.-S. performed the molecular dynamics simulations. J.L. performed the HPLC analyses. W.L. and R.-T.G. collected crystallographic data and solved all of the structures. J.F. carried the docking experiments. X.L. optimized the fermentation conditions. X.C., H.Z., M.A.M.-S., S.O., Q.W. and D.Z. wrote the manuscript. S.O., Q.W., Y.M and D.Z. directed the project.
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Supplementary information
Supplementary Information
Supplementary Methods, Tables 1–4, Figs. 1–28 and References
Supplementary Data 1
Initial_structure_MD_F12-C_1a-13R17S
Supplementary Data 2
Initial_structure_MD_F12-C_3k-2R3S
Supplementary Data 3
Initial_structure_MD_F12-C_3k-2S3R
Supplementary Data 4
Initial_structure_MD_F12-D_1a-13R17R
Supplementary Data 5
Initial_structure_MD_WT
Supplementary Data 6
Initial_structure_MD_WT-E_1a-13R17R
Supplementary Data 7
Initial_structure_MD_WT-E_1a-13R17S
Supplementary Data 8
Initial_structure_MD_WT-E_1a-13S17R
Supplementary Data 9
Initial_structure_MD_WT-E_1a-13S17S
Supplementary Data 10
Initial_structute_MD_F12
Supplementary Data 11
Substrate_1a_QM
Supplementary Data 12
Substrate_3k_QM
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Chen, X., Zhang, H., Maria-Solano, M.A. et al. Efficient reductive desymmetrization of bulky 1,3-cyclodiketones enabled by structure-guided directed evolution of a carbonyl reductase. Nat Catal 2, 931–941 (2019). https://doi.org/10.1038/s41929-019-0347-y
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DOI: https://doi.org/10.1038/s41929-019-0347-y
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