Letter | Published:

Accessing non-natural reactivity by irradiating nicotinamide-dependent enzymes with light

Nature volume 540, pages 414417 (15 December 2016) | Download Citation

Abstract

Enzymes are ideal for use in asymmetric catalysis by the chemical industry, because their chemical compositions can be tailored to a specific substrate and selectivity pattern while providing efficiencies and selectivities that surpass those of classical synthetic methods1. However, enzymes are limited to reactions that are found in nature and, as such, facilitate fewer types of transformation than do other forms of catalysis2. Thus, a longstanding challenge in the field of biologically mediated catalysis has been to develop enzymes with new catalytic functions3. Here we describe a method for achieving catalytic promiscuity that uses the photoexcited state of nicotinamide co-factors (molecules that assist enzyme-mediated catalysis). Under irradiation with visible light, the nicotinamide-dependent enzyme known as ketoreductase can be transformed from a carbonyl reductase into an initiator of radical species and a chiral source of hydrogen atoms. We demonstrate this new reactivity through a highly enantioselective radical dehalogenation of lactones—a challenging transformation for small-molecule catalysts4,5,6,7. Mechanistic experiments support the theory that a radical species acts as an intermediate in this reaction, with NADH and NADPH (the reduced forms of nicotinamide adenine nucleotide and nicotinamide adenine dinucleotide phosphate, respectively) serving as both a photoreductant and the source of hydrogen atoms. To our knowledge, this method represents the first example of photo-induced enzyme promiscuity, and highlights the potential for accessing new reactivity from existing enzymes simply by using the excited states of common biological co-factors. This represents a departure from existing light-driven biocatalytic techniques, which are typically explored in the context of co-factor regeneration8,9.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    et al. Engineering the third wave of biocatalysis. Nature 485, 185–194 (2012)

  2. 2.

    & in Enzyme Catalysis in Organic Synthesis, Ch. 41 (eds , & ) 1695–1723 (Wiley VCH, 2012)

  3. 3.

    & Chemomimetic biocatalysis: exploiting the synthetic potential of cofactor-dependent enzymes to create new catalysts. J. Am. Chem. Soc. 137, 13992–14006 (2015)

  4. 4.

    , & Enantioselective hydrogen transfer from a chiral tin hydride to a prochiral carbon-centered radical. Angew. Chem. Int. Edn 36, 235–236 (1997)

  5. 5.

    & Enantioselective radical reactions. Top. Curr. Chem. 263, 107–162 (2006)

  6. 6.

    Asymmetric catalysis activated by visible light. Chem. Commun. (Camb.) 51, 3290–3301 (2015)

  7. 7.

    Radical mechanisms of enzymatic catalysis. Annu. Rev. Biochem. 70, 121–148 (2001)

  8. 8.

    , & Photobiocatalysis: the power of combining photocatalysis and enzymes. Chemistry 21, 10940–10959 (2015)

  9. 9.

    et al. Cofactor-free light-driven whole-cell cytochrome P450 catalysis. Angew. Chem. Int. Edn 54, 969–973 (2015)

  10. 10.

    , & Photochemistry of flavoprotein light sensors. Nat. Chem. Biol. 10, 801–809 (2014)

  11. 11.

    & Light-dependent protochlorophyllide oxidoreductase: phylogeny, regulation, and catalytic properties. Biochemistry 54, 5255–5262 (2015)

  12. 12.

    , & Visible light photoredox catalysis with transition metal complexes: applications in organic synthesis. Chem. Rev. 113, 5322–5363 (2013)

  13. 13.

    , , & Preparation, characterization, and oxygenase activity of a photocatalytic artificial enzyme. ChemBioChem 16, 1880–1883 (2015)

  14. 14.

    , & Photoreduction of alkyl halides by an NADH model compound. an electron transfer chain mechanism. J. Am. Chem. Soc. 105, 4722–4727 (1983)

  15. 15.

    , & Photoinduced mechanisms of electron-transfer oxidation of NADH analogues and chemiluminescence. Detection of the keto and enol radical cations. J. Am. Chem. Soc. 125, 4808–4816 (2003)

  16. 16.

    , , , & Selective debromination and α-hydroxylation of α-bromo ketones using Hantzsch esters as photoreductants. Adv. Synth. Catal. 358, 74–80 (2016)

  17. 17.

    , , & Catalytic hydrogenation of α,β-epoxy ketones to form β-hydroxyketones mediated by an NADH coenzyme model. Org. Lett. 8, 3449–3451 (2006)

  18. 18.

    et al. Determination of the C4-H bond dissociation energies of NADH models and their radical cations in acetonitrile. Chemistry 9, 871–880 (2003)

  19. 19.

    , & Electron-transfer photoredox catalysis: development of a tin-free reductive dehalogenation reaction. J. Am. Chem. Soc. 131, 8756–8757 (2009)

  20. 20.

    & Photochemical and chemical enzyme catalyzed debromination of meso-1,2-dibromostilbene in multiphase systems. J. Am. Chem. Soc. 108, 1080–1082 (1986)

  21. 21.

    , & Practical chiral alcohol manufacture using ketoreductases. Curr. Opin. Chem. Biol. 14, 1–8 (2009)

  22. 22.

    et al. Access to lactone building blocks via horse liver alcohol dehydrogenase-catalyzed oxidative lactonization. ACS Catal. 3, 2436–2439 (2013)

  23. 23.

    , , , & Structure of a NADPH-dependent blue fluorescent protein revealed the unique role of Gly176 on the fluorescence enhancement. J. Struct. Biol. 174, 485–493 (2011)

  24. 24.

    Reduction of acetophenone to R(+)-phenylethanol by a new alcohol dehydrogenase from Lactobacillus kefir. Appl. Microbiol. Biotechnol. 34, 15–19 (1990)

  25. 25.

    et al. Origins of stereoselectivity in evolved ketoreductases. Proc. Natl Acad. Sci. USA 112, E7065–E7072 (2015)

  26. 26.

    , , , & The crystal structure of R-specific alcohol dehydrogenase from Lactobacillus brevis suggests the structural basis of its metal dependency. J. Mol. Biol. 327, 317–328 (2003)

  27. 27.

    et al. Structures of alcohol dehydrogenases from Ralstonia and Sphingobium spp. reveal the molecular basis for their recognition of ‘bulky–bulky’ ketones. Top. Catal. 57, 356–365 (2014)

  28. 28.

    & AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J. Comput. Chem. 31, 455–461 (2010)

  29. 29.

    , & Structural biology of the aldo-keto reductase family of enzymes: catalysis and cofactor binding. Cell Biochem. Biophys. 38, 79–101 (2003)

  30. 30.

    et al. Access to enantiopure α-alkyl-β-hydroxy esters through dynamic kinetic resolutions employing purified/overexpressed alcohol dehydrogenases. Adv. Synth. Catal. 354, 1743–1749 (2012)

Download references

Acknowledgements

Financial support was provided by Princeton University. D.G.O. also acknowledges financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC). We thank the MacMillan group for use of their chiral high-performance liquid-chromatography and cyclic-voltammetry equipment; G. Scholes for providing the time-resolved fluorescence instrument; H. Yayla of the Knowles group for assistance with cyclic-voltammetry experiments; B. Shields of the Doyle group and the Scholes Group for collection of the LED emission spectrum; and G. Huisman of Codexis for conversations regarding the nature of the mutants in the Codexis KRED kit.

Author information

Affiliations

  1. Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA

    • Megan A. Emmanuel
    • , Norman R. Greenberg
    • , Daniel G. Oblinsky
    •  & Todd K. Hyster

Authors

  1. Search for Megan A. Emmanuel in:

  2. Search for Norman R. Greenberg in:

  3. Search for Daniel G. Oblinsky in:

  4. Search for Todd K. Hyster in:

Contributions

M.A.E and T.K.H. designed the experiments, performed and analysed experiments, and prepared the manuscript. N.R.G. performed and analysed experiments. D.G.O. collected and analysed time-resolved fluorescence data.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Todd K. Hyster.

Reviewer Information

Nature thanks E. Meggers and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Methods, Supplementary Text and Data, Supplementary Figures 1-10 and additional references (see Contents for more details).

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature20569

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.