Asymmetric bioreduction of alkenes using ene-reductases YersER and KYE1 and effects of organic solvents

Article abstract of DOI:10.1021/ol200394p

Asymmetric trans-bioreduction of activated alkenes by KYE1 from Kluyveromyces lactis and Yers-ER from Yersinia bercovieri, two ene-reductases from the Old Yellow Enzyme family, showed a broad substrate spectrum with a moderate to excellent degree of stereoselectivity. Both substrate- and enzyme-based stereocontrols were observed to furnish opposite stereoisomeric products. The effects of organic solvents on enzyme activity and stereoselectivity were outlined in this study, where two-phase systems hexane and toluene are shown to sustain bioreduction efficiency even at high organic solvent content.

Full text of DOI:10.1021/ol200394p

ORGANIC
LETTERS
2011
Vol. 13, No. 10
2540–2543
Asymmetric Bioreduction of Alkenes
Using EneÀReductases YersER and
KYE1 and Effects of Organic Solvents
†
‡
†
Yanto Yanto, Christoph K. Winkler, Stephanie Lohr, Melanie Hall,†,‡ Kurt Faber,‡
ꢀ
and Andreas S. Bommarius*,†
School of Chemical and Biomolecular Engineering, Parker H. Petit Institute of
Bioengineering and Biosciences, Georgia Institute of Technology, 315 Ferst Drive,
Atlanta, Georgia 30332-0363, United States, and Department of Chemistry, Organic and
Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
andreas.bommarius@chbe.gatech.edu
Received March 7, 2011
ABSTRACT
Asymmetric trans-bioreduction of activated alkenes by KYE1 from Kluyveromyces lactis and Yers-ER from Yersinia bercovieri, two
eneÀreductases from the Old Yellow Enzyme family, showed a broad substrate spectrum with a moderate to excellent degree of
stereoselectivity. Both substrate- and enzyme-based stereocontrols were observed to furnish opposite stereoisomeric products. The effects
of organic solvents on enzyme activity and stereoselectivity were outlined in this study, where two-phase systems hexane and toluene are shown
to sustain bioreduction efficiency even at high organic solvent content.
The application of biocatalyst has become an increas-
ingly attractive route in the production of enantiomerically
pure compounds in industry due to exquisite regio- and
stereoselectivity.1 One of the emerging platforms is the
asymmetric reduction of activated CdC bonds due to the
potential to create up to two new stereogenic centers in the
process.2 EneÀreductases from the Old Yellow Enzyme
(OYE) family have been reported to catalyze the reduction
of a variety of different substrates such as R,β-unsaturated
compounds (activated by electron-withdrawing groups
such as aldehydes, ketones, imides, nitro groups, nitriles,
carboxylic acids, esters) and nitro derivatives such as nitro
esters and nitro aromatics.3 The reduction reaction cata-
lyzed by the OYE family of flavoproteins proceeds in a
strict trans fashion, with hydride attack derived from the
† Georgia Institute of Technology.
‡ University of Graz.
(1) (a) Muller, M. Curr. Opin. Biotechnol. 2004, 15, 591. (b) Zaks, A.
Curr. Opin. Chem. Biol. 2001, 5, 130.
(2) (a) Hall, M. ; Yanto, Y.; Bommarius, A. S. Encyclopedia on
Industrial Biotechnology; Wiley: Hoboken, NJ, 2010; pp 2234À2247. (b)
Toogood, H. S.; Gardiner, J. M.; Scrutton, N. S ChemCatChem 2010, 2,
892. (c) Stuermer, R.; Hauer, B.; Hall, M.; Faber, K. Curr. Opin. Chem.
Biol. 2007, 11, 203. (d) Toogood, H. S.; Fryszkowska, A.; Hare, V.;
Fisher, K.; Roujeinikova, A.; Leys, D.; Gardiner, J. M.; Stephens,
G. M.; Scrutton, N. S. Adv. Synth. Catal. 2008, 350, 2789. (e) Hall,
M.; Stueckler, C.; Kroutil, W.; Macheroux, P.; Faber, K. Angew. Chem.,
Int. Ed. Engl. 2007, 46, 3934.
(3) (a) Williams, R. E.; Rathbone, D. A.; Scrutton, N. S.; Bruce, N. C.
Appl. Environ. Microbiol. 2004, 70, 3566. (b) Kosjek, B.; Fleitz, F. J.;
Dormer, P. G.; Kuethe., J. T.; Devine, P. N. Tetrahedron: Asymmetry
2008, 19, 1403. (c) Yanto, Y.; Hall, M.; Bommarius, A. S. Org. Biomol.
Chem. 2010, 8, 1826. (d) Hall, M.; Stueckler, C.; Ehammer, H.; Pointner,
E.; Oberdorfer, G.; Gruber, K.; Hauer, B.; Stuermer, R.; Kroutil, W.;
Macheroux, P.; Faber, K. Adv. Synth. Catal. 2008, 350, 411.
r
10.1021/ol200394p
Published on Web 04/21/2011
2011 American Chemical Society

Table 1. Asymmetric Bioreduction Study Using EneÀReductases KYE1 and YersER
a nc: no conversion.
nicotinamide cofactor.4 Utilizing the coupled-enzyme ap-
proach, anefficient cofactorrecyclingsystemwithenzymes
such as glucose dehydrogenase (GDH) is commonly ap-
plied to decrease the high costs of redox cofactor for larger
scale synthesis.5,11a Furthermore, a light-driven cofactor
regeneration system6 and a nicotinamide-independent re-
duction system7 were recently reported to yield high
substrate conversion and product enantiopurity, again
highlighting the potential of OYEs in industrial biotech-
nological applications.
In a previous work, we began to characterize three
eneÀreductases: KYE1 from Kluyveromyces lactis, Yers-
ER from Yersinia bercovieri, and XenA from Pseudomonas
putida. We have shown that these three eneÀreductases
feature broad but different substrate specificity on various
R,β-unsaturated carbonyl compounds. XenA was further
demonstrated to possess moderate to excellent stereoselectiv-
ity and catalyze the reduction of various nitro compounds.8
In this study, we further explore the catalytic efficiency and
stereoselectivity of YersER and KYE1 with a new set of
substrates including R,β-alkyl-substituted cyclic enones, enol
(4) (a) Stott, K.; Saito, K.; Thiele, D. J.; Massey, V. J. Biol. Chem.
1993, 268, 6097. (b) Vaz, A. D.; Chakraborty, S.; Massey, V. Biochem-
istry 1995, 34, 4246.
(5) (a) Muller, A.; Hauer, B.; Rosche, B. Biotechnol. Bioeng. 2007, 98,
22. (b) Swiderska, M. A.; Stewart, J. D. Org. Lett. 2006, 8, 6131. (c) Hall,
M.; Stueckler, C.; Kroutil, W.; Macheroux, P.; Faber, K. Angew. Chem.,
Int. Ed. Engl. 2007, 46, 3934.
(6) Taglieber, A.; Schulz, F.; Hollmann, F.; Rusek, M.; Reetz, M. T.
Chembiochem. 2008, 9, 565.
(7) Stueckler, C.; Reiter, T. C.; Baudendistel, N.; Faber, K. Tetra-
hedron 2010, 66, 663.
(8) (a) Chaparro-Riggers, J. F.; Rogers, T. A.; Vazquez-Figueroa, E.;
Polizzi, K. M.; Bommarius, A. S. Adv. Synth. Catal. 2007, 349, 1521. (b)
Yanto, Y.; Yu, H. H.; Hall, M.; Bommarius, A. S. Chem. Commun. 2010,
8809.
Org. Lett., Vol. 13, No. 10, 2011
2541

ethers, carboxylic esters, nitrostyrenes, and maleimide-type
compounds and test both enzymes in aqueousÀorganic
media.
organic solvents (both water miscible and immiscible), ar-
ranged according to denaturation capacity scale,13 were
tested with KYE1 and YersER for the bioreduction of
2-cyclohexen-1-one in 20% organic solvent/buffer system
(Table 2). Screening results showed that both KYE1 and
YersER retained highest enzyme activity in ethylene glycol,
dimethyl sulfoxide (DMSO), and in the hexaneÀ and
tolueneÀbuffer systems. Given the low level of amino acid
identity and similarity between KYE1 and YersER (36% and
53%, respectively), the result also suggests that, in this case,
the effect of organic solvents on enzyme stability is indepen-
dent of both the denaturing capacity scale and the protein
amino acid sequences. The experimentally determined
threshold concentration (C50), at which half inactivation of
the enzyme is observed, for both KYE1 and YersER was
43.3% for ethylene glycol/buffer and 27.5% for dimethyl
sulfoxide/buffer (see the Supporting Information). For the
two-phase hexane and toluene systems, the catalytic reaction
does not seem to be impaired even at 70% organic solvent
level, corresponding to a phase ratio of organic to aqueous
solvent of 2.33 (0.7:0.3) (detailed results in the Supporting
Information). Since both hexane and toluene are immiscible
with water, the enzymes are expected to be in the aqueous
phase with minimum contact of organic solvents and thus
retained full enzyme activity. However, there is only one
phase for ethylene glycol and DMSO systems; therefore, the
enzymes were much more exposed to the organic solvent,
which resulted in decreased activity. A similar trend was
observed for the bioreduction of citral with KYE1 and
The substrate study for both KYE1 and YersER were
performed over 12 h with the GDH cofactor recycling
system (Table 1, detailed experimental procedures available
in Supporting Information). Both enzymes showed excel-
lent stereoselectivity toward the reduction of R,β-alkyl-
substituted cyclic enones (1À4) and predominantly furn-
ished the corresponding (S)-products. Substrate-based
stereocontrol was observed for the reduction of R,β-sub-
stituted enol ethers.9 The reduction of five-membered cyclic
enol ethers (5 and 6) furnished the corresponding (S)-
products, but upon increasing the ring size to cyclohexeno-
neÀenol ether (8), the stereopreference switched to (R) for
both enzymes; furthermore, by changing the side-chain
substitution from a methyl group (8) to a sterically more
demanding n-propyl (10) or phenyl group (9), the substrate
was flipped in the enzyme-binding pockets to furnish the
corresponding (S)-products. The results from substrates
1À13 showed that KYE1 and YersER strongly prefer R-
substituted substrates, and significantly lower activity was
observed for β-substituted substrates (2, 4, 7, 11, and 13).
The conversion of dicarboxylic esters, nitrostyrenes, mal-
eimide, and citral (14À21) revealed a moderate to high
degree of enzymatic activity together with excellent product
enantiopurity, except for 19, where a racemic product was
observed for both enzymes.2c Enzyme-based stereocontrol
was again seen in the reduction of 15 and 21, where KYE1
and YersER formed the corresponding (R)- and (S)-pro-
ducts, respectively.10 These results show that prediction of
reaction stereo-outcome proves difficult. While KYE1 and
YersER share 36% amino acid identity and structures
among OYE enzymes are highly conserved, a pair of major
residues involved in substrate binding varies from one
enzyme to the other (KYE: H191/N194; YersER: H173/
H176). Similar variations occur within the OYE family and
may be involved in substrate recognition/orientation.2b
To enhance the technological utility of eneÀreductase for
gram-scale synthesis, especially for less water-soluble sub-
strates, we investigated the effects of organic solvents on
catalytic efficiency and stereoselectivity. To date, there are
few data published addressing solvent effects on OYEs,11 and
this work represents the first detailed study of organic solvent
effects on eneÀreductase. Water/organic binary co-solvent
systems often enhance the catalytic properties of enzymes and
potentially offer significant advantages for increasing bioca-
talyst performance in synthetic chemistry.12 Twelve different
Table 2. Catalytic Activity for Bioreduction of 2-Cyclohexen-1-
one in 20% Organic Solvent System
conv (%)
solvent
DCa
log Pb
YersER
KYE1
ethylene glycol
methanol
18.7
30.5
38.8
54.4
60.3
63.3
64.3
78.2
92.1
100
À1.43
À0.74
À0.74
À1.35
À0.32
À1.35
À0.32
3.5
100
61.8
3.1
98.9
23.7
3.2
1,2-propanediol
ethanol
6.9
6.6
dimethyl sulfoxide
dimethylformamide
acetonitrile
100
5.1
98.5
4.1
0.6
0.2
acetone
9.8
5.3
1,4-dioxane
À0.27
0.46
10.7
1.0
9.1
tetrahydrofuran
toluene
0.7
137.9
144.4
2.46
98.1
94.8
97.8
95.3
hexane
3.5
a DC: denaturing capacity. b log P: partition coefficient.
(9) Winkler, C. K.; Stueckler, C.; Mueller, N. J.; Pressnitz, D.; Faber,
K. Eur. J. Org. Chem. 2010, 33, 6354. The numbering presented in
Scheme 1 of this reference was erroneous; correct labeling should assign
the alkoxy-functionalized cyclohexen-2-ones with 1aÀ4a and 7a and
cyclopenten-2-ones with 5a, 6a and 8a
(10) Stueckler, C.; Hall, M.; Ehammer, H.; Pointner, E.; Kroutil, W.;
Macheroux, P.; Faber, K. Org. Lett. 2007, 9, 5409.
(11) (a) Bougioukou, D. J.; Walton, A. Z.; Stewart, J. D. Chem.
Commun. 2010, 8558. (b) Stueckler, C.; Mueller, N. J.; Winkler, C. K.;
Glueck, S. M.; Gruber, K.; Steinkellner, G.; Faber, K. Dalton. Trans.
2010, 39, 8472.
YersER, where the conversion and product enantiopurity
were monitored in the presence of 0 À 50% organic solvent
(Figure 1). Surprisingly, the product enantiopurity decreased
tracking the diminishing enzyme activity (lower conversion)
for ethylene glycol (Figure 1a) and DMSO (Figure 1b). The
hexaneÀ and tolueneÀbuffer systems again showed constant
(12) (a) Fitzpatrick, P. A.; Klibanov, A. M. J. Am. Chem. Soc. 1991,
113, 3161. (b) Klibanov, A. M. Nature 2001, 409, 241.
(13) Khmelnitsky, Y. L.; Mozhaev, V. V.; Belova, A. B.; Sergeeva,
M. V.; Martinek, K. Eur. J. Biochem. 1991, 198, 31.
2542
Org. Lett., Vol. 13, No. 10, 2011

Figure 1. Dependence of organic solvent concentration on the catalytic activity and stereoselectivity of KYE1 and YersER in the
asymmetric bioreduction of citral (21).
enzymatic activity and product enantiopurity in the investi-
gated phase ratios (Figure 1c,d).
efforts should focus on predictability and effective enhance-
ment of product enantiopurity.
In conclusion, KYE1 and YersER displayed both sub-
strate-based (enol ethers, diesters) and enzyme-based (citral,
lactones) stereocontrol. A biphasic organic solvent/buffer
system revealed no effect of the phase ratio (in the range of
0À2.3) on catalytic efficiency or stereoselectivity. In mono-
phasic DMSOÀ and ethylene glycolÀbuffer systems, the
activity declined markedly beyond 20% and 35% solvent,
respectively, and stereoselectivity of citral bioconversion
declined as well with rising solvent content. To further
enhance the applicability of eneÀreductases, future research
Acknowledgment. The experimental assistance of Cle-
mens Stueckler (University of Graz) and Hua-Hsiang Yu
(Georgia Institute of Technology) is gratefully acknow-
ledged.
Supporting Information Available. General synthesis
and analytical methods, with additional results. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Org. Lett., Vol. 13, No. 10, 2011
2543