Abstract
Despite their intriguing photophysical and photochemical activities, naturally occurring photoenzymes have not yet been repurposed for new-to-nature activities. Here we engineered fatty acid photodecarboxylases to catalyse unnatural photoredox radical C–C bond formation by leveraging the strongly oxidizing excited-state flavoquinone cofactor. Through genome mining, rational engineering and directed evolution, we developed a panel of radical photocyclases to facilitate decarboxylative radical cyclization with excellent chemo-, enantio- and diastereoselectivities. Our high-throughput experimental workflow allowed for the directed evolution of fatty acid photodecarboxylases. An orthogonal set of radical photocyclases was engineered to access all four possible stereoisomers of the stereochemical dyad, affording fully diastereo- and enantiodivergent biotransformations in asymmetric radical biocatalysis. Molecular dynamics simulations show that our evolved radical photocyclases allow near-attack conformations to be easily accessed, enabling chemoselective radical cyclization. The development of stereoselective radical photocyclases provides unnatural C–C-bond-forming activities in natural photoenzyme families, which can be used to tame the stereochemistry of free-radical-mediated reactions.
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Acknowledgements
This research is supported by the National Institutes of Health (NIH; R35GM128779 to P.L., R35GM147387 to Y.Y.), American Chemical Society Petroleum Research Fund (ACS PRF; 65807-DNI1) and the National Science Foundation (NSF; OCE-1756947 to D.L.V.). We acknowledge the NSF BioPACIFIC Materials Innovation Platform (MIP; DMR-1933487) and NSF Materials Research Science and Engineering Centers (MRSEC) at University of California, Santa Barbara (UCSB; DMR-2308708) for access to instrumentation. Computational studies were carried out at the University of Pittsburgh Center for Research Computing and the Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support (ACCESS) programme, supported by NSF award numbers OAC-2117681 and OAC-2138259. We thank Y. Wang for critical reading of this manuscript.
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Y.Y. conceived and directed the project. S.J. performed all the enzyme engineering and substrate scope studies. S.J. and A.V.-E. performed enzyme mining and molecular cloning with Y.Y. and D.L.V., and J.W. provided guidance. D.L., S.J. and X.L. synthesized all the substrates and racemic products for analysis. B.K.M. carried out the computational studies with P.L. providing guidance. Y.Y., P.L., S.J. and B.K.M. wrote the manuscript with the input of all other authors.
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Y.Y. and S.J. are inventors on a patent application submitted by the University of California Santa Barbara (UC case no. 2024-843) that covers compositions, methods and applications of evolved RAPs derived from natural FAPs. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Origin of stereoconvergent photobiocatalytic radical cyclisation with (E)- and (Z)-1a.
a, Stereoconvergent photobiotransformation of (E)-1a and (Z)-1a. b, Photoisomerisation of (Z)-1a with FAD (free cofactor). c, Initial rate and photoisomerisation with CvRAP1.
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Supplementary Figs. 1–6, Discussion and Tables 1–26.
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Ju, S., Li, D., Mai, B.K. et al. Stereodivergent photobiocatalytic radical cyclization through the repurposing and directed evolution of fatty acid photodecarboxylases. Nat. Chem. (2024). https://doi.org/10.1038/s41557-024-01494-0
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DOI: https://doi.org/10.1038/s41557-024-01494-0