Nicotinamide-dependent Ene reductases as alternative biocatalysts for the reduction of activated alkenes

Article abstract of DOI:10.1002/ejoc.201200776

Four NAD(P)H-dependent non-flavin ene reductases have been investigated for their ability to reduce activated C=C bonds in an asymmetric fashion by using 20 structurally diverse substrates. In comparison with flavin-dependent Old Yellow Enzyme homologues, a higher degree of electronic activation was required, because the best activities were obtained with enals and nitroalkenes rather than enones and carboxylic esters. Although FaEO from Fragaria x ananassa (strawberry) and its homologue SlEO from Solanum lycopersicum (tomato) exhibited a narrow substrate spectrum, progesterone 5β-reductase (At5β-StR) from Arabidopsis thaliana (thale cress) and leukotriene B₄ 12-hydroxydehydrogenase (LTB₄DH/PGR) from Rattus norvegicus (rat) appear to be promising candidates, in particular for the asymmetric bioreduction of open-chain enals, nitroalkenes and α,β-unsaturated γ-butyrolactones. Competing nitro reduction and non-enzymatic Weitz-Scheffer epoxidation were largely suppressed. Electronically activated alkenes have been stereoselectively reduced by using a single-enzyme-cofactor system employing nicotinamide-dependent non-flavin ene reductases. Copyright

Full text of DOI:10.1002/ejoc.201200776

Job/Unit: O20776
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Date: 30-07-12 11:52:12
Pages: 7
FULL PAPER
DOI: 10.1002/ejoc.201200776
Nicotinamide-Dependent Ene Reductases as Alternative Biocatalysts for the
Reduction of Activated Alkenes
[
a]
[b]
[b]
[c]
Katharina Durchschein, Silvia Wallner, Peter Macheroux, Wilfried Schwab,
[d]
[e]
[a]
Thorsten Winkler, Wolfgang Kreis, and Kurt Faber*
Keywords: Alkenes / Reduction / Biotransformations / Enzymes
Four NAD(P)H-dependent non-flavin ene reductases have
been investigated for their ability to reduce activated C=C
bonds in an asymmetric fashion by using 20 structurally di-
sicum (tomato) exhibited a narrow substrate spectrum, pro-
gesterone 5β-reductase (At5β-StR) from Arabidopsis thaliana
4
(thale cress) and leukotriene B 12-hydroxydehydrogenase
verse substrates. In comparison with flavin-dependent Old
Yellow Enzyme homologues, a higher degree of electronic
activation was required, because the best activities were ob-
tained with enals and nitroalkenes rather than enones and
carboxylic esters. Although FaEO from Fragaria x ananassa
(LTB DH/PGR) from Rattus norvegicus (rat) appear to be
4
promising candidates, in particular for the asymmetric biore-
duction of open-chain enals, nitroalkenes and α,β-unsatu-
rated γ-butyrolactones. Competing nitro reduction and non-
enzymatic Weitz–Scheffer epoxidation were largely sup-
pressed.
(strawberry) and its homologue SlEO from Solanum lycoper-
mon fold, which determines their stereopreference.[4] In the
search for a more simple single-enzyme-cofactor alternative,
Introduction
The asymmetric bioreduction of electronically activated
alkenes, such as enones, enals, α,β-unsaturated carboxylic
acids (or derivatives thereof) and nitroalkenes, has been in-
tensively investigated during the past few years by using fla-
we turned our attention to flavin-independent ene re-
[1,5]
ductases. In contrast to the large number of OYEs,
only
a handful of NAD(P)H-dependent ene reductases have
been described: little is known about their mechanism, and
even less about their substrate scope, which determines their
synthetic applicability. Most of the non-flavo ene reductases
[
1]
voproteins from the “Old Yellow Enzyme” (OYE) family.
These enzymes depend on flavin mononucleotide (FMN)
as a cofactor, which launches a nucleophilic (Michael-type)
attack of a hydride from flavin-N5 onto Cβ; a proton is
delivered onto Cα from the opposite side leading to an anti
addition of hydrogen.[ Since the oxidised flavin regains its
hydride from NAD(P)H,[ the overall system becomes quite
complex, as it depends on two enzymes, the ene-reductase
and an NAD(P)H-recycling enzyme, and two cofactors,
FMN and NAD(P)H. Furthermore, all OYEs share a com-
[6]
[7–14]
have been found in plants and mammalian tissues.
Two NAD(P)H-dependent ene reductases, (+)-pulegone
and (–)-isopiperitenone reductase, play a central role in
monoterpene biosynthesis in Mentha piperita (peppermint).
Although the latter enzyme stereoselectively acts on the en-
docyclic C=C bond of (–)-isopiperitenone to furnish (+)-
cis-isopulegone, (+)-pulegone reductase reduces the exocy-
clic double bond of (+)-pulegone in a non-stereoselective
fashion to yield a mixture of (+)-isomenthone and (–)-
menthone. Both enzymes show high substrate specificity.[
2]
3]
15]
[
a] Department of Chemistry, Organic & Bioorganic Chemistry,
(
+)-Pulegone reductase belongs to the NADP-dependent
leukotriene B dehydrogenase family. A member of this
enzyme group, leukotriene B4 12-hydroxydehydrogenase
LTB DH/PGR), showed typical alcohol dehydrogenase ac-
University of Graz,
Heinrichstrasse 28, 8010 Graz, Austria
4
Fax: +43-316-380-9840
E-mail: Kurt.Faber@Uni-Graz.at
(
Homepage: http://biocatalysis.uni-graz.at/
b] Institute of Biochemistry, Technical University of Graz,
Petersgasse 12/II, 8010 Graz, Austria
c] Biotechnology of Natural Products, Technical University
München,
4
[
[
tivity on the C12 hydroxy group in LTB , although later
4
work reassessed it as 15-oxoprostaglandin 13-reductase due
to its ability to reduce the C13 C=C double bond conju-
gated to the C15 carbonyl group in 15-oxo-prostaglandin
and 15-oxo-lipoxin A4, which classifies it as an ene re-
Arcisstrasse 21, 80333 München, Germany
d] Institute of Molecular Enzyme Technology, University of
Düsseldorf, Research Centre Jülich,
Stetternicher Forst, 52426 Jülich, Germany
e] Department of Biology, University Erlangen-Nürnberg,
Staudtstrasse 5, 91058 Erlangen, Germany
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200776.
[
[
ductase. Hence, this protein is able to catalyse both a 1,2-
[7,14]
and a 1,4-addition of a hydride onto an enone.
Various
[
8]
homologues of LTB DH/PGR are found in man, Rattus
4
norvegicus (rat), Sus scrofa (wild boar), Bos taurus (cow),
Eur. J. Org. Chem. 0000, 0–0
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K. Faber et al.
FULL PAPER
[
11,14]
and Arabidopsis thaliana (Scheme 1, Table 1).[18,27] It is known that LTB DH/PGR
Cavia porcellus (guinea pig)
4
[
16]
[18]
(
thale cress).
The best-investigated isozyme was isolated shows a higher preference towards NADPH, whereas for
and displays broad reductive activity on alk- At5β-StR, NADPH is the only co-substrate that cannot be
[
17]
from rat
enals and alkenones.[
17,18]
Similar conjugate reduction ac- replaced by NADH.
[27]
By using FaEO/SlEO, both cofac-
tivities were found with P1 ζ-crystallin from Arabidopsis tors are accepted, but (for kinetic reasons) better results
[
24]
thaliana (P1-ZCr), an oxidative-stress-induced 2-alkenal re- were obtained by using NADPH.
ductase, which catalyses the reduction of toxic 2-alkenals
[
19]
and 3-alken-2-ones,
pounds.
o-quinones and azo carbonyl com-
[
19,20]
These azo compounds were reduced at the
N=N bond to yield the corresponding hydrazine deriva-
tives. Surprisingly, LTB DH/PGR and P1-ZCr do not re-
4
duce cyclohexenone, a typical lead substrate for OYEs. Al-
though OYE homologues follow a strict anti addition of
hydrogen, LTB DH/PGR mediated both anti and syn ad-
4
dition of [2H] depending on the substrate stereochemistry,
which was proven by the bioreduction of both enantiomers
of perillaldehyde, which furnished the cis product in a
stereoconvergent fashion.[ A related case of syn addition
Scheme 1. Asymmetric bioreduction of activated alkenes by using
nicotinamide-dependent ene reductases.
21]
has been reported for a Nicotiana tabacum (tobacco) re-
Surprisingly, 2-cyclohexenone (1a), which is a classic test
ductase acting on verbenone and carvone.[ A novel enone compound for OYEs, was poorly accepted by non-flavin
22]
oxidoreductase, which was putatively assigned as quinone ene reductases. It was not accepted by LTB DH/PGR,
4
oxidoreductase from Fragaria x ananassa (strawberry) FaEO or SlEO, regardless of the cofactor (NADH or
[
23]
FaEO, and its homologues SlEO from Solanum lycoper- NADPH) or recycling system (glucose dehydrogenase/gluc-
+
sicon var. cerasiforme (tomato) and AcQR from Ananas ose/NAD or glucose 6-phosphate dehydrogenase/glucose
+
comosus (pineapple) were recently studied for their activities 6-phosphate/NADP ) used. Only At5β-StR was active and
on exocyclic conjugated C=C bonds of 4-hydroxy-5-methyl- showed a preference for NADPH over NADH (conv. Ͼ99
2
-methylene-3(2H)-furanone (HMMF) and derivatives vs. 31%).
[
23,24]
thereof.
Nicotinamide-dependent steroid reductases
α-Substituted cyclic enones 2a, 3a and 4a were asymmet-
from plants are known to reduce activated C=C bonds in rically reduced by At5β-StR; the conversion and stereo-
hormones, such as progesterone, but may display relaxed selectivity were enhanced with increasing ring size and ste-
substrate specificity, as shown with Δ4,5-steroid 5β-re- ric hindrance, with (R)-levodione (3b) being formed in up
[
28]
ductase (At5β-StR) from Arabidopsis thaliana, which also to 92% conversion and 45% ee.
A contrasting picture
accepts non-steroidal enones and acrylate esters.[
25]
was observed with (R)- or (S)-pulegone (5a); 5a was a non-
To evaluate the applicability of these enzymes for the substrate for At5β-StR, FaEO and SlEO, but was reduced
bioreduction of activated alkenes, we examined the sub- by LTB DH/PGR, albeit with low diastereoselectivity
4
strate scope and stereoselectivity of four ene reductases (75–86% conv., de Յ 5%). (2E)-2-Ethylidene-4-hydroxy-5-
from different sources: FaEO from Fragaria x ananassa methyl-3(2H)-furanone (EDHMF; 6a) is a derivative of
[
23]
(
strawberry), its homologue SlEO from Solanum lycoper- HMMF [4-hydroxy-5-methyl-2-methylene-3(2H)-furanone],
[
24]
sicum (tomato),
progesterone 5β-reductase (At5β-StR) the precursor for furaneol [4-hydroxy-2,5-dimethyl-3(2H)-
from Arabidopsis thaliana (thale cress)[ and leukotriene B4 furanone, HDMF], the aroma component in strawberries,
25]
1
2-hydroxydehydrogenase (LTB DH/PGR) from Rattus and was assumed to be the “natural” substrate for FaEO
4
[
21]
norvegicus (rat).
According to their amino acid se- and SlEO. As a result of the high instability of HMMF, we
quences, these enzymes are not related to each other, as decided to synthesize EDHMF, which needs not be
[
24]
expressed by the low degree of similarity/identity (FaEO trapped. Although 6a was indeed fully converted by both
and At5β-StR show 9% identity/13% similarity, FaEO and FaEO and SlEO, a strong reaction in the absence of enzyme
LTB DH/PGR show 23% identity/39% similarity and was also observed (85% conv.) under standard conditions.
4
At5β-StR and LTB DH/PGR have 6% identity/12% simi- Hence, NADPH alone is evidently able to reduce the highly
4
[
26]
larity).
activated conjugated cyclic enol ether 6a without significant
catalytic assistance of FaEO and SlEO. To overcome the
apparent modest degree of activation of enones, enals were
tested: although the simple cyclohexenecarbaldehyde (7a)
Results and Discussion
was reasonably well reduced by LTB DH/PGR and At5β-
4
To obtain a broad picture of the substrate tolerance of StR in up to 45% conversion, the sterically more hindered
the four enzyme candidates in comparison to that of flavin- (S)-perillaldehyde [(S)-8a] was fully reduced by LTB4DH/
dependent OYE-type ene reductases, we chose a broad PGR to 8b with a preference for the (Z) isomer (cis 36%),
range of α/β-unsaturated ketones, aldehydes, carboxylic while At5β-StR showed even stronger preference for the (Z)
acid derivatives, such as esters, a cyclic imide, lactones and isomer (cis 48%). The long-chain enal citral (9a) was the
nitroalkenes as substrates using NADPH as cofactor best substrate for At5β-StR, which furnished the olfactory
2
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Ene Reductases for the Reduction of Activated Alkenes
Table 1. Asymmetric bioreduction of activated alkenes 1a–20a by NADPH-dependent ene reductases LTB
4
DH/PGR, At5β-StR, FaEO
[
a]
and SlEO.
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K. Faber et al.
FULL PAPER
Table 1. (Continued).
[
(
(
a] Reaction conditions: substrate (10 mm), ene reductase (protein concentration 125–135 μg/mL), NADPH (15 mm), Tris-HCl buffer
+
50 mm, pH = 7.5), 24 h, 30 °C, 120 rpm; n.d. = not determined; n.a. = not applicable; n.c. = no conversion. [b] GDH (10 U)/NAD
+
+
100 μm)/Glu (20 mm). [c] GDH(10 U)/NADP (100 μm)/Glu (20 mm). [d] G6PDH(10 U)/NADP (100 μm)/Glu6P (20 mm). [e] Back-
ground reaction with NADPH (in the absence of enzyme) shows 85% conversion. [f] A trace of primary alcohol originating from
competing C=O bioreduction was detected (Յ1%). [g] As a result of the low conversion, the ee value may be prone to error.
agent (S)-citronellal (9b) with Ͼ99% conversion and excel- pentenone, cyclohexenone (1a) and ketoisophorone (3a) as
lent stereoselectivity (Ͼ99% ee).
substrates (see the Supporting Information), which are de-
Among the carboxylic acid derivatives, regioisomeric rived from non-enzymatic Weitz–Scheffer epoxidation by
α,β-unsaturated diesters 10a–12a were reduced to 10b with H O produced by enzyme-catalyzed O2 oxidation of
2
2
modest conversion (conv.max. 18% from 10a), in up to 50% NAD(P)H. This side-reaction was dominant with several
ee for (S)-10b obtained from 11a (11% conversion). A sim- OYE homologues, such as YqjM, as well as flavin-depend-
ilar picture was observed with methyl 2-(hydroxymethyl)- ent quinone reductases and NADH:FMN oxidoreductases,
acrylate (13a, “Roche ester”),[
29]
which gave (S)-13b in such as Lot6P and YcnD, respectively. Side-reactions of
[32]
modest 28% ee with LTB DH/PGR. N-Substituted male- flavoproteins involving nitro group reduction to yield ni-
4
imides are highly activated and hence are usually well ac- troso compounds, which may undergo bio-Nef degrada-
[
33]
cepted by OYEs, predominantly yielding the (R)-configured tion
product.
or spontaneously cyclise to form oxazete deriva-
[
5,30]
[34]
Among the non-flavo ene reductases, only tives, were not detected with substrates 18a–20a.
At5β-StR behaved in this manner to form (R)-14b in 67%
The overall picture of the substrate tolerance of non-
conversion and 93% ee, the other enzymes being inactive. flavo ene reductases shows significant differences to that of
To extend the substrate portfolio, α,β-unsaturated butyro- OYEs. (1) Of the four enzyme candidates, FaEO and SlEO
lactones were tested. Despite the usefulness of α-chiral lac- exhibit a very narrow substrate spectrum. In contrast,
tones as synthons, this type of substrate has scarcely been LTB DH/PGR and At5β-StR appear more suitable for the
4
investigated by using OYEs.[ Although the unsubstituted asymmetric bioreduction of non-natural compounds. (2) To
31]
test compound 15a and the α-methyl analogue 16a were make a conjugated C=C bond amenable to bioreduction, a
poorly converted by LTB DH/PGR (ca. 2% conv.), the α- higher degree of electronic activation (as judged by the elec-
4
methylene derivative 17a was transformed into (R)-16b with tron-withdrawing properties of the respective electron-with-
8
4% conversion and good stereoselectivity (70% ee) Note drawing group) is required. Hence, typical OYE substrates,
that the electronically more activated isosteric cyclopen- such as enones and α,β-unsaturated (di)esters, were only po-
tenone 4a was relatively slowly reduced. orly converted, whereas enals, maleimide and nitroalkenes
Finally, the most highly activated substrates, nitroalk- were good substrates. (3) Surprisingly, sterically demanding
enes, turned out to be well accepted by all enzymes: unsub- substrates were often better accepted than “slim” counter-
stituted nitrocyclohexene (18a) was fully converted into the parts (except for At5β-StR), which is in contrast to the ten-
[
1,27,35]
saturated product 18b and open-chain nitrostyrene ana- dency observed with OYEs.
(4) exo-Methylene C=C
logues 19a and 20a gave high conversions (up to 95%) and bonds were better accepted than endo analogues, which
good stereoselectivities (up to 90% ee) for the correspond- again is contrary to the preference of OYEs.[36] (5) Pro-
ing nitroalkanes 19b and 20b. An interesting switch in miscuous catalytic activities observed with OYEs, such as
[
32]
[33]
stereopreference between LTB DH/PGR (R) and At5β-StR NAD(P)H oxidation by O2
or nitro group reduction,
were either marginal or completely absent. (6) α,β-Unsatu-
With FaEO and SlEO, minor amounts of epoxide (Յ7%, rated γ-butyrolactones proved to be a promising new class
corrected for background value) were observed with cyclo- of substrates for conjugated bioreduction.
4
(S) was observed.
4
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Ene Reductases for the Reduction of Activated Alkenes
Table 2. Optimized reaction conditions using different recycling systems.[a]
[
a] Reaction conditions: Substrate (10 mm), ene reductase (125–135 μg/mL protein concentration), Tris-HCl buffer (50 mm, pH = 7.5),
+
NAD(P)H (20 mm) or NAD(P) (100 μm), glucose dehydrogenase (GDH) or glucose 6-phosphate dehydrogenase (G6PDH, 10 U), glucose
Glu, 20 mm) or glucose 6-phosphate (Glu6P, 20 mm), 24 h, 30 °C, 120 rpm. [b] As a result of the low conversion, the ee value may be
(
prone to error.
Cofactor Dependence and Recycling
obtained with enals and nitroalkenes rather than enones
and carboxylic esters. Although FaEO from Fragaria x an-
anassa (strawberry) and its homologue SlEO from Solanum
lycopersicum (tomato) appear less useful for synthetic bio-
transformations due to their narrow substrate spectrum,
progesterone 5β-reductase (At5β-StR) from Arabidopsis
The type of cofactor and its respective recycling system
have been shown to influence the reaction rate and (to a
lesser extent) also the stereochemistry[ of flavin-depend-
ent OYEs, which operate by a bi-bi ping-pong mecha-
30]
[
2,37]
nism,
that is, NAD(P)H delivers its hydride onto the
thaliana (thale cress) and leukotriene B 12-hydroxydehy-
4
flavin (“reductive half reaction”), and then the substrate en-
ters and the product leaves the active site (“oxidative half
reaction”). To investigate whether non-flavo ene reductases
show related effects, ketoisophorone (3a) and the α-methyl-
ene lactone 17a were tested by using NAD(P)H in the pres-
ence of the cofactor recycling systems (Table 2). No signifi-
cant differences between the types of cofactors were ob-
drogenase (LTB DH/PGR) from Rattus norvegicus (rat) are
4
promising candidates, in particular for the asymmetric bio-
reduction of open-chain enals (such as citral), nitroalkenes
and α,β-unsaturated γ-butyrolactones.
Experimental Section
served with FaEO and SlEO. Although the activity of Standard Procedure for Enzymatic Bioreduction: An aliquot of en-
LTB DH/PGR and At5β-StR remained unchanged with zyme [FaEO, SlEO, LTB DH/PRG-1 (protein concentration
4
4
3
a, the ee of 3b could be enhanced from 45 to 88% when 125 μg/mL) or At5β-StR (protein concentration 135 μg/mL)] was
using the GDH/NADP /Glu cofactor recycling system in-
+
added to a Tris-HCl buffer solution (0.8 mL, 50 mm, pH = 7.5)
containing the substrate (10 mm) and the cofactor NAD(P)H
stead of NADPH alone. An even stronger influence was
(
15 mm). The mixture was shaken at 30 °C and 120 rpm. After 24 h,
the products were extracted with EtOAc (2ϫ0.5 mL). The com-
bined organic phases were dried with Na SO and analysed by
observed with LTB DH/PGR, which strongly preferred
4
NADPH over NADH by a factor of about 20,[ and goes
in hand with significantly enhanced stereoselectivity with α-
methylene-γ-butyrolactone (17a; ee for 16b was 40–88%).
18]
2
4
achiral GC to determine the conversion and by chiral GC or
HPLC, respectively, to determine the enantiomeric excess. For all
biotransformations, one flavin-dependent ene reductase (OYEs 1–
These effects are intriguing, because LTB DH/PGR is
4
[
18]
known to proceed by a Theorell–Chance mechanism,
3) was used as a positive control and to determine the absolute
that is, both substrate and cofactor are bound in the active configuration.[
1,29,30,36]
+
site with NADPH binding first and NADP leaving last.
Standard Procedure for Cofactor Recycling: An aliquot of enzyme
Preliminary data indicate that the presence of organic co- (protein conc. as above) was added to a Tris-HCl buffer solution
solvents had no beneficial effects (see the Supporting Infor- (0.8 mL, 50 mm, pH = 7.5) containing the substrate (10 mm), the
oxidized form of the cofactor (NAD+ or NADP , 100 μm), the co-
substrate (glucose or glucose-6-phosphate, 20 mm) and the corre-
sponding recycling enzyme (glucose dehydrogenase, or glucose-6-
phosphate dehydrogenase, 10 U). The mixture was shaken at 30 °C
and 120 rpm for 24 h and worked up as described above. For all
+
mation).
Conclusions
The activities of the four non-flavin ene reductases inves- biotransformations, one flavin-dependent ene reductase (OYEs 1–
tigated in this study indicate that these NAD(P)H-depend- 3) was used as a positive control.[
1,29,30,36]
ent enzymes require a higher degree of electronic activation Supporting Information (see footnote on the first page of this arti-
than the OYE homologues, because the best activities were cle): The source of materials, chemicals and enzymes, synthesis of
Eur. J. Org. Chem. 0000, 0–0
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K. Faber et al.
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substrates and reference materials, determination of absolute con-
figuration, chiral and non-chiral analytical methods, additional
data on co-solvent studies and Weitz–Scheffer epoxidation.
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[
[
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Published Online: ᭿
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20776
/KAP1
Date: 30-07-12 11:52:12
Pages: 7
Ene Reductases for the Reduction of Activated Alkenes
Alkene Reduction
K. Durchschein, S. Wallner, P. Macheroux,
W. Schwab, T. Winkler, W. Kreis,
K. Faber* .......................................... 1–7
Nicotinamide-Dependent Ene Reductases
as Alternative Biocatalysts for the Re-
duction of Activated Alkenes
Electronically activated alkenes have been
stereoselectively reduced by using a single-
enzyme-cofactor system employing nicotin-
amide-dependent non-flavin ene re-
ductases.
Keywords: Alkenes / Reduction / Biotrans-
formations / Enzymes
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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