Cofactor-Free, Direct Photoactivation of Enoate Reductases for the Asymmetric Reduction of C=C Bonds

Article abstract of DOI:10.1002/anie.201702461

Enoate reductases from the family of old yellow enzymes (OYEs) can catalyze stereoselective trans-hydrogenation of activated C=C bonds. Their application is limited by the necessity for a continuous supply of redox equivalents such as nicotinamide cofactors [NAD(P)H]. Visible light-driven activation of OYEs through NAD(P)H-free, direct transfer of photoexcited electrons from xanthene dyes to the prosthetic flavin moiety is reported. Spectroscopic and electrochemical analyses verified spontaneous association of rose bengal and its derivatives with OYEs. Illumination of a white light-emitting-diode triggered photoreduction of OYEs by xanthene dyes, which facilitated the enantioselective reduction of C=C bonds in the absence of NADH. The photoenzymatic conversion of 2-methylcyclohexenone resulted in enantiopure (ee>99 %) (R)-2-methylcyclohexanone with conversion yields as high as 80–90 %. The turnover frequency was significantly affected by the substitution of halogen atoms in xanthene dyes.

Full text of DOI:10.1002/anie.201702461

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Accepted Article
Title: Cofactor-Free, Direct Photoactivation of Enoate Reductases for
Asymmetric Reduction of C=C Bonds
Authors: Sahng Ha Lee, Da Som Choi, Milja Pesic, Yang Woo,
Caroline Paul, Frank Hollmann, and Chan Beum Park
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201702461
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10.1002/anie.201702461
Angewandte Chemie International Edition
((will be filled in by the editorial staff))
Photobiocatalysis
Cofactor-Free, Direct Photoactivation of Enoate Reductases for
Asymmetric Reduction of C=C Bonds**
Sahng Ha Lee, Da Som Choi, Milja Pesic, Yang Woo Lee, Caroline E. Paul, Frank Hollmann* and Chan Beum
Park*
Abstract: Enoate reductases from the family of Old Yellow
Enzymes (OYEs) can catalyze stereoselective trans-hydrogenation of
activated C=C bonds. Despite their potential, however, their
application is limited by the necessity for continuous supply of redox
equivalents such as nicotinamide cofactors [NAD(P)H]. Here, we
report visible light-driven activation of OYEs through NAD(P)H-
free, direct transfer of photoexcited electrons from xanthene dyes to
the prosthetic flavin moiety. Our spectroscopic and electrochemical
analyses verified spontaneous association of rose bengal and its
derivatives with OYEs. Illumination of a white light-emitting-diode
triggered photoreduction of OYEs by xanthene dyes, which
facilitated the enantioselective reduction of C=C bonds in the
absence of NADH. The photoenzymatic conversion of 2-
methylcyclohexenone resulted in enantiopure (ee >99%) (R)-2-
methylcyclohenanone with conversion yields as high as 80-90%.
Scheme 1. Schematic illustration of the light-driven activation of flavin-
containing TsOYE using rose bengal (RB) as a photosensitizer. Photoexcitation
of the molecular photosensitizer reduces the active species of TsOYE to
The turnover frequency was significantly affected by the substitution
of halogen atoms in xanthene dyes. The NADH-free, xanthene-
sensitized photobiocatalytic platform was successfully applied to
different homologues of OYEs from Thermus scotoductus and
Bacillus subtilis. This work demonstrates a simple and versatile way
of activating OYEs by direct coupling of OYE-catalysis with
molecular photocatalysis.
catalyze an enantioselective reduction of 2-methlycyclohexenone to 2-
methylcyclohexanone.
majority of OYE applications have been made by mimicking the
biological scheme through in situ regeneration of reduced
nicotinamide
cofactors
using
enzymatic,[4]
chemical,[5]
electrochemical,[6] and photochemical[7] approaches.
Here, we report NAD(P)H-free activation of flavin-
containing OYEs by molecular photosensitizers that allow for direct
transfer of photoinduced electrons to the prosthetic flavin moiety.
Rose bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein, RB)
and its xanthene derivatives are explored as photosensitizers to drive
OYE-catalyzed redox reactions under illumination with light-
emitting-diodes (LEDs). Xanthene dyes are low cost
photosensitizers, capable of capturing visible light energy with high
quantum efficiency, which have been applied to reductive chemical
reactions such as solar hydrogen evolution as well as photochemical
NADH regeneration[8] and for the reduction of metalloenzymes (e.g.,
heme-containing cytochrome P450s, metal cluster-containing
hydrogenases).[9] Scheme 1 illustrates direct transfer of photoexcited
electrons from RB to a flavin-containing OYE homologue from
Thermus scotoductus (TsOYE)[10] upon visible light irradiation with
triethanolamine (TEOA) as an electron donor. Successive transfer of
electrons reduces the enzyme-bound FMN moiety and drives the
enantioselective conversion of 2-methylcyclohexenone into (R)-2-
methylcyclohexanone.
Typically, OYEs are reduced by NAD(P)H that donate two
electrons and one proton directly to the enzyme-bound FMN moiety
(Figure 1A). A step-wise reduction by successive transfer of
electron yields a single-electron reduced semiquinone state of FMN,
which can be fully reduced by taking an additional electron.[11] We
investigated the possibility of photosensitized reduction of TsOYE
by RB using UV-Vis spectroscopy. TsOYE shows an absorbance
peak at 464 nm due to a strong π – π* transition associated with the
oxidized redox state of the enzyme-bound FMN. Any observed
decrease of the intensity can be attributed to the reduction of FMN
to FMNH- through the step-wise transfer of electrons and
protonation.[12] According to our result (Figure 1B), the absorbance
peak at 464 nm decreased gradually during light irradiation in the
presence of both RB and TEOA, which we attribute to the reduction
of the prosthetic FMN by photoinduced electron transfer from RB to
1
E
nzyme-catalyzed transformations provide unmatched high
efficiency, regio-, stereo-, and enantio-selectivities under mild
conditions.[1] Enoate reductases from the family of Old Yellow
Enzymes (OYEs) are a class of flavin-containing enzymes that
catalyze asymmetric reduction of activated (or conjugated) C=C
bonds, producing industrially useful chiral alkanes.[2] The redox
enzymes contain a prosthetic group of flavin mononucleotide
(FMN), which, in its reduced form (FMNH-), transfers a hydride by
a Michael-type reaction to the conjugated C=C double bond. The
regeneration of the reduced flavin occurs by the transfer of two
electrons and two protons,[3] which is achieved in vivo through the
mediation of reduced nicotinamide cofactors [NAD(P)H]. For the
development of biocatalytic processes, however, NAD(P)H is too
expensive to be supplied in a stoichiometric amount; thus, the
Dr. S. H. Lee, D. S. Choi, Y. W. Lee, and Prof. Dr. C. B. Park
Department of Materials Science and Engineering,
Korea Advanced Institute of Science and Technology
335 Science Road, Daejeon 305-701, Republic of Korea
E-mail: parkcb@kaist.ac.kr
Dr. C. E. Paul, Dr. M. Pesic, and Prof. Dr. F. Hollmann
Department of Biotechnology, Delft University of Technology,
Van der Maasweg 9, 2629 HZ Delft, The Netherlands.
E-mail: f.hollmann@tudelft.nl
[] Corresponding authors.
[] This work was supported by the National Research
Foundation (NRF) via the Creative Research Initiative Center
(Grant number: NRF-2015 R1A3A2066191), Republic of
Korea, and the Netherlands Organisation for Scientific
Research by a VICI grant (Grant number: 724.014.003).
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/anie.201xxxxxx.
This article is protected by copyright. All rights reserved.

10.1002/anie.201702461
Angewandte Chemie International Edition
photocurrent (ca. 1.2 μA) was generated in the presence of both
TsOYE and RB upon light illumination, which is attributed to the
decrease in the number of electrons from the electrode to the
enzyme due to the photosensitization of RB. In contrast, there was
no photoresponse with either TsOYE or RB alone. Taken together,
the spectroscopic, electrochemical, and photochemical results
suggest a spontaneous association between TsOYE and RB through
a direct energy transfer relationship, facilitating RB-sensitized
reduction of TsOYE.
Next, we investigated the asymmetric reduction of conjugated
C=C double bonds via light-driven activation of TsOYE. As shown
in Figure S4, we observed
a gradual conversion of 2-
methylcyclohexenone to enantiopure (R)-2-methycyclohexanone (ee
> 99%) with 76% yield after illumination of white LED (4.74
mW/cm2) for 150 min. A series of control experiments in the
absence of each reaction component (i.e, light, electron donor,
photosensitizer, and enzyme) revealed that only trace amounts (~0.2
mM at maximum) of enantiopure product was detected without light,
TEOA, or RB (see Figure S5A), and no product was formed in the
absence of TsOYE. This result indicates that photosensitization is
essential for generating high energy electrons to initiate
photoinduced electron transfer for OYE catalysis and, to sustain
light-driven
enzymatic
turnover,
both
light-harvesting
photosensitizer (i.e., RB) and electron-supplying agent (i.e., TEOA)
are required. We also observed a clear dependency of initial reaction
rate and product yield on the intensity of white LED light (Figure
S5B).o elucidate the rate-determining factor in the photobiocatalytic
reaction, we measured changes in the initial rate and product yield
according to the concentrations of TsOYE and RB, respectively. As
shown in Figure S6A, proportional enhancements of reaction rate
and product yield were observed with the increasing concentration
of RB (from 0 to 100 μM) at a fixed TsOYE concentration (18 μM).
The initial turnover frequency (TOF) of TsOYE was calculated to be
256.1 ± 4.9 h-1, which was saturated with RB concentration at over
100 μM. The TOF value is comparable to those of previously
reported methyl viologen-mediated, Ru-polypyridyl-sensitized OYE
reactions.[17] The apparent saturation kinetics, which levels off at
Figure 1. A) Possible steps of enzyme-bound FMN reduction. B) UV-vis
absorbance spectra of TsOYE-RB mixture under visible light irradiation. C) The
change in the absorbance of TsOYE at 464 nm with or without RB or TEOA. An
aqueous solution containing 60 μM TsOYE, 2 μM RB, 5 mM CaCl2, and 200
mM TEOA was irradiated by a 450 W Xenon lamp equipped with 420 nm cut-off
filter.
TsOYE with TEOA as an electron donor. Note that there was
negligible change in the absorbance of TsOYE without either RB or
TEOA even under illumination (Figure 1C). The photoreduction of
OYE-bound FMN by RB was further confirmed by using another
OYE homologue from Bacillus subtilis (YqjM)[13] (Figure S1).
According to our result, three isosbestic points at 318 nm, 382nm
and 407 nm were clearly observed during the photoreduction of
prosthetic FMN by RB, which indicates that the semiquinone was
built up as an intermediate during the step-wise reduction process.
In addition, we observed spontaneous association of RB with
TsOYE by measuring the changes in absorbance and
photoluminescence spectra of RB upon addition of TsOYE. The
absorbance of RB at 546 nm gradually red-shifted with the
increasing concentration of TsOYE (Figure 2A), which indicates a
change of RB’s ionization state upon its binding to the enzyme.[14]
The change in the absorbance at around 464 nm was caused by the
increasing concentration of TsOYE. Furthermore, we observed that
the addition of TsOYE induces quenching and red-shift of RB’s
photoluminescence spectrum (see Figure S2), which is attributed to
fluorescence energy transfer from RB to TsOYE.
higher RB concentration, indicate
a Michaelis-Menten-type
behavior of TsOYE with RB as a co-substrate, instead of NADH. In
a control experiment conducted for comparison using NADH as a
cosubstrate(see Figure S6B), we found that the reaction rate
saturated at around 2 mM NADH and TOF was estimated to be 734
We further investigated electrochemical relationship between
RB and TsOYE using protein film voltammetry. For the analysis of
TsOYE's electrochemical behavior, we prepared a TsOYE-coated
glassy carbon disk electrode by using didodecyldimethylammonium
bromide as a support for protein adsorption.[15] We observed TsOYE
reduction at around -0.5 V (vs. Ag/AgCl) (Figure 2B), which
corresponds to the reduction of enzyme-bound flavin cofactor
according to the literature.[16] In the presence of RB, we detected a
negative shift of approximately 60 mV in the reduction potential of
TsOYE. Considering that reduction potential of RB exists at a more
negative potential (~ -0.92 V),[8b] this result suggests that TsOYE
and RB form an electrochemically hybridized species that exhibits a
co-reducing property due to the binding affinity between the two
components. Upon the addition of RB, TsOYE showed apparent
increases in the reduction peak current in the presence of 2-
methylcyclohexenone, a substrate of TsOYE, indicating that the
association of TsOYE with RB does not alter its catalytic activity
(see Figure S3). We also observed a distinguishable feature of
photochemical activity resulted by the co-existence of TsOYE and
RB (Figure 2C); at an applied potential of -0.6 V, an anodic
Figure 2. A) Spectrophotometric changes in the absorbance of RB upon
addition of TsOYE. B) Cyclic voltammograms of TsOYE modified electrode in
the presence or absence of RB. C) Photocurrent response of TsOYE, RB, and
TsOYE with RB at an applied potential of -0.6 V (vs. Ag/AgCl). D) Proposed
mechanism of electron transfer from RB to TsOYE upon visible-light irradiation
in the presence of sacrificial electron donor (TEOA)..
2
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10.1002/anie.201702461
Angewandte Chemie International Edition
Table 1. Reduction of 2-methylcyclohexenone (R)-2-methycyclohexanone by
TsOYE in NAD(P)H-free, light-driven biocatalytic platform employing different
molecular photosensitizers.
Table 2. Photoenzymatic reduction of 2-methylcycloexenone and
cinnamaldehyde by different OYEs (TsOYE and YqjM).
[RB]
Yield[a]
[%]
[a]
Substrate
OYE
TTNOYE
eea
Photosensitizer
(PS)
TOF[a]
[h-1]
Yield[b]
[%]
ee[b]
[%]
[μM]
[b]
[b]
TTNPS
TTNOYE
TsOYE
TsOYE
YqjM
80
160
80
169
254
149
282
120
115
77
51
76
44
84
36
34
23
>99 (R)
Eosin Y
Erythrosine B
Phloxine B
Rose Bengal
Fluorescein
Rhodamine B
Rhodamine 6G
FMN
118
100
78
66.5
73.9
45.5
53.1
9.7
106
118
73
295
328
202
235
43
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
YqjM
160
80
>99 (R)
52
85
TsOYE
TsOYE
YqjM
-
-
-
4.6
0
15
160
80
1.1
1.8
1.7
6.5
5
YqjM
160
106
32
-
2.4
4.8
1.1
4.9
18
[a]Determined by GC analysis after 180 min reaction. Reaction conditions: 24
μM [OYE], 10 mM CaCl2, 8 mM substrates in a 200 mM TEOA buffer pH 7.5
4.1
[a]Turnover frequency of photosensitizers determined after 30 min.
1
[b]Determined by GC analysis after 180 min reaction. ee: enantiomeric excess,
Reaction conditions: 50 μM [PS], 18 μM [TsOYE], 10 mM CaCl2, 8 mM 2-
methylchclohexenone in a 200 mM TEOA buffer, pH 7.5
and a product yield of 9.7%. We attribute the low photocatalytic
activities of non-halogenated derivatives (i.e., fluorescein,
rhodamines) to their higher fluorescence quantum yields (e.g.,
Фfluorescein, rhodamine B ~0.95) than those of other halogenated species
(e.g., ФEY = 0.57)[20], which causes a shorter life time of excited
± 18 h-1 at maximum. The stoichiometric ratio of TsOYE and RB
(1:5) at the saturation was two-order lower than that of TsOYE and
NADH (1:333), indicating high efficiency of RB for the activation
TsOYE. We found that the reaction rate reaches a saturation at
around the RB/TsOYE ratio of approximately five, regardless of the
TsOYE concentration, indicating that the formation of RB/TsOYE
complex is critical for driving the photobiocatalytic reaction with a
threshold ratio of approximately five (see Figure S7). When we
varied TsOYE concentration at a fixed amount of RB (60 μM) (see
Figure S8A), the reaction showed a maximum rate of ~2.92 mM h-1
with 12 μM TsOYE. A decrease in the reaction rate was observed at
the TsOYE concentration higher than 12 μM, which we attribute to
the drastic decrease in the RB/TsOYE ratio from 5 to 1.6 (see
Figure S8B). A reduced association of RB per TsOYE is expected
because the molar ratio of RB to TsOYE decreases with the
increasing TsOYE concentration at a fixed RB concentration.
Figure 2D illustrates an energy diagram for photoinduced electron
transfer from RB to TsOYE. Upon visible-light absorption, RB
promotes an internal conversion of its electrons from a ground state
(HOMORB ~ 0.75 V vs. Ag/AgCl) to the excited state (LUMORB ~ -
1.17 V).[18] The excited electrons in RB possess enough potential
energy to drive the reduction of TsOYE-bound FMN (Ereduction ~ -
0.43 V), and the oxidation of TEOA (Eoxidation ~ 0.86 V) stabilizes
RB to sustain its photocatalytic turnover. The repeated transfer of
photoexcited electrons from RB to TsOYE fully reduces enzyme-
bound FMN to FMNH-, driving catalytic cycle for asymmetric C=C
reduction.
triplet state due to the high fluorescence. According to the
literature,[8b,
halogen substitution enhances the singlet-triplet
21]
transition state of aromatic molecules by increasing spin-orbit
mixing, and the substitution of heavier halogen atom makes the
molecule easier to associate with other species due to the decrease in
the electronegativity. In the case of FMN, we observed a very low
TOF and product yield, comparable to that of fluorescein, which
should be caused by high reactivity of photoexcited flavins with
dissolved oxygen, producing reactive radical species that denature
enzymes; thus, the use of free FMN as a photocatalyst requires
deaeration of the reaction medium to remove dissolved oxygen and
drive substrate conversion.[22] In contrast, the photoreaction carried
out by RB was not affected under O2-rich conditions; our
experiments conducted with three different ratios (2, 5, 10) of
RB/TsOYE under O2-purged or N2-purged conditions also confirms
that excessive RB over TsOYE under O2-rich conditions had
negligible effect on RB-mediated reductions (see Figure S9).
According to the literature,[23] a nonspecific binding of RB with
protein induces static quenching of the excited state of RB that
prevents photosensitized production of singlet oxygen. The
spontaneous association of RB with TsOYE should facilitate the
reduction of enzyme-bound FMN, instead of singlet oxygen
production. Furthermore, the fast reduction of RB in the presence of
TEOA would have prevented the generation of singlet oxygen by
transforming the singlet-excited RB to its reduced state.
We confirmed a broad applicability of the NADH-free,
xanthene-sensitized photobiocatalytic platform by extending it to
YqjM (Table 2). The catalytic reaction by YqjM showed a product
yield of 84% with a TTN of 282, which is comparable to the result
with TsOYE (yield 76%, TTN 254). The catalytic performances of
both enzymes were also dependent on the concentration of RB.
Furthermore, we performed RB-sensitized photobiocatalytic
reduction of α,β-unsaturated aldehyde (i.e., cinnamaldehyde) by
TsOYE and YqjM. Consensus to the reaction with 2-
methylcyclohexenone, both enzymes showed apparent activity
towards the reduction of the unsaturated aldehyde with a product
yield around 30%. The lower catalytic activity compared to 2-
methylcyclohexenone is attributed to the inhibitory effect of the
product on OYE and poor solubility of the substrate in the aqueous
medium,[17] which should be improved by applying a biphasic
reaction system in future studies. Overall, the results show that the
xanthene-sensitized photoactivation can be applied to broader types
of OYEs for different substrates.
We further investigated different xanthene molecules and
natural flavin mononucleotide (FMN) for direct photoactivation of
TsOYE. According to our result (Table 1), halogenated derivatives
of xanthenes such as eosin Y (EY), erythrosine B (ErB), phloxine B
(PhB), and RB showed high activity in TsOYE-catalyzed reduction
of 2-methylcyclohexenone; EY exhibited best performance in terms
of reaction rate with a TOF of 118 and ErB resulted in the highest
conversion yield (73.9%). Our result indicates that the substitution
of a heavier halogen atom in xanthenes resulted in slower reaction
rate (i.e., TOFs in the order of EY > ErB > PhB > RB). We ascribe
the result to an increased non-specific association of halogenated
xanthene dyes to TsOYE, considering that hydrophobic interaction
of xanthenes with proteins is known to increase with their
halogenations in the order of EY < ErB < RB.[19] On the other hand,
non-halogenated derivatives of xanthene dyes, such as fluorescein,
rhodamine B, and rhodamine 6G, showed significantly lower–by
more than one order–performance compared to halogenated
xanthenes. For example, fluorescein showed an initial TOF of 4.6 h-
3
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10.1002/anie.201702461
Angewandte Chemie International Edition
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showed their co-reducing behavior with strong photoelectrochemical
responses. The RB-sensitized photoenzymatic conversion of 2-
methylcyclohexenone resulted in
a maximum 90% yield of
enantiopure (R)-2-methycyclohexanone (ee > 99%) with a TOF of
256 h-1. A decreasing order of photoenzymatic activity was observed
with the increasing weight of substituted halogen atoms in
xanthenes. A general applicability of the NADH-free, RB-sensitized
photobiocatalytic system was demonstrated by using different OYE
homologues (e.g., TsOYE, YqjM) and substrates (e.g., 2-
methylcyclohexenone, cinnamaldehyde). We anticipate our strategy
would provide a simple and cost-effective platform for developing
scalable and sustainable biocatalytic processes utilizing light energy.
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Keywords: redox enzymes · photocatalysis · asymmetric reduction ·
enoate reductases · green chemistry
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This article is protected by copyright. All rights reserved.

10.1002/anie.201702461
Angewandte Chemie International Edition
Photobiocatalysis
Sahng Ha Lee, Da Som Choi, Milja
Pesic, Yang Woo Lee, Caroline E.
Paul, Frank Hollmann, and Chan
Beum Park
__________ Page – Page
Cofactor-Free, Direct Photoactivation
of Enoate Reductases for Asymmetric
Reduction of C=C Bonds
Flavin-containing Old Yellow Enzymes (OYE) are activated by molecular photosensitizers
through direct transfer of photoinduced electrons to the prosthetic flavin moiety without any
NAD(P)H cofactor. Rose bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein, RB) and its
xanthene derivatives are explored as photosensitizers to drive OYE-catalyzed reduction of
conjugated C=C bonds under visible light illumination. The RB-sensitized photoenzymatic
reduction of 2-methylcyclohexenone resulted in a high conversion yield of enantiopure (R)-2-
methycyclohexanone (ee > 99%) with turnover frequency comparable to the reaction
mediated by natural NAD(P)H cofactors.
5
This article is protected by copyright. All rights reserved.