Letter reSeArCH
10. Conrad, K. S., Manahan, C. C. & Crane, B. R. Photochemistry of flavoprotein
light sensors. Nat. Chem. Biol. 10, 801–809 (2014).
11. Gabruk, M. & Myslia-Kurdziel, B. Light-dependent protochlorophyllide
oxidoreductase: phylogeny, regulation, and catalytic properties. Biochemistry
54, 5255–5262 (2015).
12. Prier, C. K., Rankic, D. R. & MacMillan, D. W. C. Visible light photoredox catalysis
with transition metal complexes: applications in organic synthesis. Chem. Rev.
113, 5322–5363 (2013).
KRED-3 to achieve good levels of enantioselectivity (Fig. 2, compounds
17, 19 and 21).
We also carried out mechanistic experiments to further elucidate
the nuances of this reaction. When the reaction is run with deuterated
(D8) isopropyl alcohol, such that deuterated NAD(P)H is generated
in situ, deuterolactone (22) is formed predominantly (92% deute-
rium incorporation), with excellent enantioselectivity (e.r. =98/2), 13. Gu, Y., Ellis-Guardiola, K., Srivastava, P. & Lewis, J. C. Preparation,
characterization, and oxygenase activity of a photocatalytic artificial enzyme.
supporting nicotinamide’s role as the hydrogen-atom source. (Fig. 3a
and Supplementary Fig. 10). Over the course of the reaction, racemic
ChemBioChem 16, 1880–1883 (2015).
14. Fukuzumi, S., Hironaka, K. & Tanaka, T. Photoreduction of alkyl halides by an
halolactones are converted to enantioenriched product. Although the
radical species is prochiral, it is unlikely that it survives for long enough
to diffuse into the enzyme’s active site. As such, we were curious as
to whether a kinetic resolution of the starting material occurs over
the course of the reaction. Surprisingly, there appears to be very lit-
tle preference for one enantiomer of starting material over the other
an e.r. of 96/4 and 99/1, respectively, with respect to the alcohol stereo-
centre. In both cases, the alcohol stereocentre was formed selectively as
the (R)-isomer, matching the observed selectivity in the dehalogenation
reaction. These results suggest that KRED-12 cannot distinguish
between enantiomers at the α-position of lactones and cyclic ketones30.
Indeed, docking models indicate that RasADH can bind both enanti-
omers of the starting material (Supplementary Fig. 2).
NADH model compound. an electron transfer chain mechanism. J. Am. Chem.
Soc. 105, 4722–4727 (1983).
15. Fukuzumi, S., Inada, S. & Suenobu, T. 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. Jung, J., Kim, J., Park, G., You, Y. & Cho, E. J. Selective debromination and
α-hydroxylation of α-bromo ketones using Hantzsch esters as
photoreductants. Adv. Synth. Catal. 358, 74–80 (2016).
17. Xu, H.-J., Liu, Y.-C., Fu, Y. & Wu, Y.-D. Catalytic hydrogenation of α,β-epoxy
ketones to form β-hydroxyketones mediated by an NADH coenzyme model.
Org. Lett. 8, 3449–3451 (2006).
18. Zhu, X.-Q. 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. Narayanam, J. M. R., Tucker, J. W. & Stephenson, C. R. J. Electron-transfer
photoredox catalysis: development of a tin-free reductive dehalogenation
reaction. J. Am. Chem. Soc. 131, 8756–8757 (2009).
20. Maidan, R. & Willner, I. Photochemical and chemical enzyme catalyzed
debromination of meso-1,2-dibromostilbene in multiphase systems. J. Am.
Chem. Soc. 108, 1080–1082 (1986).
21. Huisman, G. W., Liang, J. & Krebber, A. Practical chiral alcohol manufacture
using ketoreductases. Curr. Opin. Chem. Biol. 14, 1–8 (2009).
22. Kara, S. et al. Access to lactone building blocks via horse liver alcohol
dehydrogenase-catalyzed oxidative lactonization. ACS Catal. 3, 2436–2439
(2013).
23. Kao, T.-H., Chen, Y., Pai, C.-H., Chang, M.-C. & Wang, A. H.-J. 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. Hummel, W. Reduction of acetophenone to R(+)-phenylethanol by a new
alcohol dehydrogenase from Lactobacillus kefir. Appl. Microbiol. Biotechnol. 34,
15–19 (1990).
25. Noey, E. L. et al. Origins of stereoselectivity in evolved ketoreductases. Proc.
Natl Acad. Sci. USA 112, E7065–E7072 (2015).
26. Niefind, K., Müller, J., Riebel, B., Hummel, W. & Schomburg, D. 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. Man, H. 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. Trott, O. & Olson, A. J. 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).
On the basis of these findings, we propose the mechanism outlined
in Fig. 3d. Irradiation of the charge-transfer complex, which comprises
halolactone (1) and NAD(P)H within the active site of KRED (I), effects
an electron transfer to form 1•−•NADPH•+⊂KRED (II, where ⊂ sym-
bolizes inclusion in the active site). This, upon mesolytic cleavage of
the C–Br bond, forms 1••NADPH•+⊂KRED (III). We hypothesize that
both enantiomers of starting material bind within the active site and,
upon dehalogenation and formation of prochiral radical 1•, undergo a
conformational change within the active site for the enantio-determin-
ing hydrogen-atom transfer, to form 2•NADP+⊂KRED (IV). Finally,
NADP+ can be reduced by either isopropyl alcohol (using native alco-
hol dehydrogenase activity) or glucose dehydrogenase to complete the
catalytic cycle.
In conclusion, we have found that photoexcitation of nicotinamide-
dependent enzymes can cause them to become catalytically promiscuous.
We anticipate that this strategy will enable many radical-mediated
reactions to be rendered highly selective.
29. Sanli, G., Dudley, J. I. & Blaber, M. Structural biology of the aldo-keto reductase
family of enzymes: catalysis and cofactor binding. Cell Biochem. Biophys. 38,
79–101 (2003).
Online Content Methods, along with any additional Extended Data display items and
these sections appear only in the online paper.
30. Cuetos, A. et al. Access to enantiopure α-alkyl-β-hydroxy esters through
dynamic kinetic resolutions employing purified/overexpressed alcohol
Data Availability The data that support the findings in this study are available from
the corresponding author upon reasonable request.
dehydrogenases. Adv. Synth. Catal.
354, 1743–1749 (2012).
Received 17 May; accepted 14 October 2016.
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.
1. Bornscheuer, U. T. et al. Engineering the third wave of biocatalysis. Nature 485,
185–194 (2012).
2. Bornscheuer, U. T. & Kazlauskas, R. J. in Enzyme Catalysis in Organic Synthesis,
Ch. 41 (eds Drauz, K., Gröger, H. & May, O.) 1695–1723 (Wiley VCH, 2012).
3. Prier, C. K. & Arnold, F. H. Chemomimetic biocatalysis: exploiting the synthetic
potential of cofactor-dependent enzymes to create new catalysts. J. Am. Chem.
Soc. 137, 13992–14006 (2015).
4. Blumenstein, M., Schwarzkopf, K. & Metzger, J. O. Enantioselective hydrogen
transfer from a chiral tin hydride to a prochiral carbon-centered radical. Angew.
Chem. Int. Edn 36, 235–236 (1997).
5. Zimmerman, J. & Sibi, M. P. Enantioselective radical reactions. Top. Curr. Chem.
263, 107–162 (2006).
6. Meggers, E. Asymmetric catalysis activated by visible light. Chem. Commun.
(Camb.) 51, 3290–3301 (2015).
7. Frey, P. A. Radical mechanisms of enzymatic catalysis. Annu. Rev. Biochem. 70,
121–148 (2001).
8. Maciá-Agulló, J. A., Corma, A. & Garcia, H. Photobiocatalysis: the power of
combining photocatalysis and enzymes. Chemistry 21, 10940–10959
(2015).
Author 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.
Author Information Reprints and permissions information is available at
paper. Correspondence and requests for materials should be addressed to
9. Park, J. H. et al. Cofactor-free light-driven whole-cell cytochrome P450
catalysis. Angew. Chem. Int. Edn 54, 969–973 (2015).
Reviewer Information Nature thanks E. Meggers and the other anonymous
reviewer(s) for their contribution to the peer review of this work.
1 5 d E c E m b E R 2 0 1 6
| V O L 5 4 0 | N A T U R E | 4 1 7
© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.