Recombinant Δ4,5-steroid 5 β-reductases as biocatalysts for the reduction of activated C=C-double bonds in monocyclic and acyclic molecules

Article abstract of DOI:10.1002/adsc.200900024

It was found that Δ^4,5-steroid 5β-reductases are capable of reducing also small molecules bearing an activated C=C double bond such as monocyclic enones and acyclic enoate esters. As preferred Δ^4,5-steroid 5β-reductase (5β-StR) for this purpose, 5β-StR from Arabidopsis thaliana was used. In part, enzyme activities are even higher than that for progesterone. Successful preliminary biotransformations with enzymatic in situ cofactor recycling were also carried out. When using the prochiral compound isophorone as a substrate, a high enantioselective reaction course (>99% ee) was observed.

Full text of DOI:10.1002/adsc.200900024

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DOI: 10.1002/adsc.200900024
Recombinant D4,5-Steroid 5b-Reductases as Biocatalysts for
the Reduction of Activated C=C-Double Bonds in Monocyclic
and Acyclic Molecules
Edyta Burda,a Marina Kraußer,a Gabriele Fischer,b Werner Hummel,c
Frieder Mꢀller-Uri,b Wolfgang Kreis,b,* and Harald Grçgera,*
a
Department of Chemistry and Pharmacy, University of Erlangen-Nuremberg, Henkestr. 42, 91054 Erlangen, Germany
Fax : (+49)-9131-85-21165; phone: (+49)-9131-85-22554; e-mail: harald.groeger@chemie.uni-erlangen.de
Department of Biology, University of Erlangen-Nuremberg, Staudtstr. 5, 91058 Erlangen, Germany
b
Fax : (+49)-9131-85-28243; phone: (+49)-9131-85-28241; e-mail: wkreis@biologie.uni-erlangen.de
Institute of Molecular Enzyme Technology at the Heinrich-Heine-University of Dꢀsseldorf, Research Centre Jꢀlich
c
Stetternicher Forst, 52426 Jꢀlich, Germany
Received: January 13, 2009; Revised: September 18, 2009; Published online: November 10, 2009
Abstract: It was found that D4,5-steroid 5b-reductas-
es are capable of reducing also small molecules
bearing an activated C=C double bond such as
monocyclic enones and acyclic enoate esters. As
preferred D4,5-steroid 5b-reductase (5b-StR) for this
purpose, 5b-StR from Arabidopsis thaliana was
used. In part, enzyme activities are even higher
than that for progesterone. Successful preliminary
biotransformations with enzymatic in situ cofactor
recycling were also carried out. When using the
prochiral compound isophorone as a substrate, a
high enantioselective reaction course (>99% ee)
was observed.
zu et al.[2a,b] and Stewart et al.[2d,4a] who applied these
enzymes in various C=C double bond reductions. In
spite of the high suitability of these enoate reductases,
there is an increasing demand for further enzymes
being capable of reducing C=C double bonds, thus ex-
panding the diversity of biocatalysts for this important
reaction. An elegant step towards this direction has
been recently reported, e.g., by Faber et al. demon-
strating the suitability of a 12-oxophytodienoate re-
ductase from tomato as an (air-stable) enoate reduc-
tase in organic syntheses.[2c,9,10] This example also
shows that enzymes of plant origin can be used as ef-
ficient biocatalysts in organic synthesis. In this context
we now communicate our preliminary results on the
suitability of D4,5-steroid 5b-reductases of plant origin
for the reduction of activated C=C double bonds in
small monocyclic and acyclic molecules.
Keywords: alkenes; biotransformations; enoate re-
ductases; enzyme catalysis; ketones; reduction
Recently, a full-length cDNA clone encoding a D4,5-
steroid 5b-reductase (At5b-StR, EC 1.1.1.145/
1.3.1.23), a member of the short-chain dehydrogenase/
The biocatalytic reduction of activated C=C double reductase (SDR) family, was isolated from Arabidop-
bonds represents an enzymatic reaction with high ap- sis thaliana leaves and expressed in Escherichia
plication potential in organic synthesis.[1] By means of coli.[11] A three-dimensional model of the enzyme
such a reaction type one can obtain access to chiral highlights its close structural similarity to the previ-
aldehydes and ketones,[2] carboxylates,[2c,3] and nitroal- ously described Digitalis lanata progesterone 5b-re-
kanes.[2c,4] However, in contrast to the reduction of ductase for which the crystal structure was deter-
C=O double bonds of ketones,[5] aldehydes,[6] and mined.[12] Referring to plant steroid reductases we
keto esters[7] with alcohol dehydrogenases, the field of were interested in investigating the suitability of these
biocatalytic C=C-reduction is still poorly explored.[8] enzymes to accept smaller molecules bearing an acti-
This is in particular due to the limited number of vated C=C double bond. For our studies we used re-
readily available and sufficiently stable recombinant combinant D4,5-steroid 5b-reductases from Digitalis
enoate reductases. The workhorses in this field are (Isoplexis) canariensis[13] and A. thaliana.[11] The latter
enoate reductases from lower fungi and bacteria,[1] enzyme shows an activity of 0.4 U per mg of protein
which show a broad substrate range, are available in for progesterone,[11] thus making it interesting for or-
recombinant form, and have been applied already in ganic synthetic purposes.
efficient reduction processes. This has been demon-
The investigation of the substrate spectrum has
strated, e.g., by impressive contributions from Shimi- been carried out by means of a spectrophotometrical
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Edyta Burda et al.
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methacrylate ester 7 (prepared via Baylis–Hillman re-
action of ethyl acrylate and formaldehyde) as a sub-
strate, an increased enzymatic activity of 154% was
observed. This result is also interesting from a syn-
thetic perspective since it shows the suitability of
Baylis–Hillman products for enzymatic enoate reduc-
tion with At5b-StR. Furthermore, this study repre-
sents one of the rare examples demonstrating the ca-
pability of steroid reductases to accept small monocy-
clic and acyclic molecules bearing a C=C double bond
in conjugation to an electron-withdrawing group as
substrates.[14] Thus, it appears that steroid reductases,
in particular progesterone reductases of plant origin,
possess a broader substrate spectrum than widely ex-
pected.
Encouraged by the positive photometer experi-
ments we subsequently studied the application of
At5b-StR from A. thaliana as biocatalyst in prepara-
tive biotransformations with in situ cofactor regenera-
tion. In situ regeneration[5] of the cofactor NADPH
(which was used in a catalytic amount) was carried
out using a glucose dehydrogenase under oxidation of
d-glucose to d-gluconolactone, thus regenerating the
required reduced form of the cofactor, NADPH. The
reduction concept with At5b-StR from A. thaliana is
shown in Table 1. When using 2-cyclohexenone (2) as
Scheme 1. Substrate spectrum of At5b-StR (for this enzyme
a specific activity of 0.4 U/mg was reported when using com-
pound 1 as a substrate, see ref.[11]).
study, measuring the consumption of the cofactor de- substrate, reduction proceeded smoothly under for-
pending on the type of substrate (according to mation of cyclohexanone with >95% conversion at
Scheme 1). We found that (in contrast to the typically 30–348C (Table 1, entry 1). This biotransformation
expected narrow substrate range of steroid-transform- underlines the suitability of this enzyme to reduce
ing enzymes) the recombinant D4,5-steroid 5b-reduc- also non-steroid-type molecules on a preparative
tase from A. thaliana (At5b-StR) shows an unexpect- scale.
edly broad substrate spectrum (Scheme 1). Notably,
A reaction at a somewhat elevated reaction temper-
small monocyclic and even acyclic substrates bearing ature of 40–418C gave similar results (entry 2). Be-
an activated C=C double bond were accepted. A high sides At5b-StR from A. thaliana we identified a D4,5-
relative enzyme activity of 559% was measured for 2- steroid 5b-reductase from Digitalis (Isoplexis) canar-
cyclohexenone (2), which exceeds significantly the iensis[13] as a further steroid reductase from a plant to
one measured for progesterone (1, 100%).
be suitable for the reduction of 2-cyclohexenone,
In contrast, for 3-methyl-substituted cyclohexe- leading to cyclohexanone with a high conversion of
nones as substrates low enzyme activities were ob- >95% as well (entry 3). It should be added that the
served with <20% for both 3-methyl-2-cyclohexenone D4,5-steroid 5b-reductase from D. (I.) canariensis was
(3) and 3,5,5-trimethyl-2-cyclohexenone (isophorone, also successfully expressed in E. coli,[13] thus being
4). The small monocyclic molecules 2 and 3 are of available in the advantageous recombinant form, too.
particular interest from a mechanistic perspective
As an example for substituted cyclohexenones, iso-
since both compounds can be considered as simple phorone (4) was used as a substrate. The reduction
monocyclic mimics of progesterone (1) and represent also proceeded, but a low conversion of 14% was ob-
substructures of 1. Thus, the large deviation of the ob- served in the presence of At5b-StR from A. thaliana
tained enzyme activities for substrates 2 and 3 is sur- as a biocatalyst (entry 4). This result is in accordance
prising and will be subject to molecular modelling with the lower enzyme activity for 4 (in comparison
studies. Furthermore, we were interested if At5b-StR with 2, see Scheme 1). This transformation of the pro-
from A. thaliana also accepts acyclic compounds, in chiral compound 4 into chiral 3,3,5-trimethyl-2-cyclo-
particular a,b-unsaturated enoate esters. However, hexanone also enabled us to study the potential of
using simple acrylate esters such as ethyl acrylate (5) At5b-StR from A. thaliana to catalyze reductions of
and ethyl methacrylate (6) resulted in low enzyme ac- activated C=C double bonds in an enantioselective
tivities of <20% and 21%, respectively. It is notewor- fashion. We were pleased to find that the reduction of
thy that the presence of a hydroxy group led to an in- 4 proceeded highly enantioselectively in the presence
crease of enzymatic activity. When using hydroxylated of At5b-StR from A. thaliana, leading to 3,3,5-tri-
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Recombinant D4,5-Steroid 5b-Reductases as Biocatalysts
Table 1. Preparative biotransformations.
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Entry[a]
R1
R2
R3
R4
T [8C]
Conversion [%]
ee [%]
1
H
H
H
H
-(CH2)3-
-(CH2)3-
-(CH2)3-
H
H
H
CH3
H
30–34
40–41
30
>95
>95
>95
14
–
–
–
2
3[b]
4
-CH2CMe2CH2-
OEt
30
>99
5
CH2OH
H
r.t.[c]
15
n.d.[d]
[a]
For general protocol, see Experimental Section; reaction time (t) was 24 h for entries 1 and 3, and 48 h for entries 2, 4 and 5.
Steroid reductase from Digitalis (Isoplexis) canariensis was used in this experiment as enoate reductase.
r.t.=room temperature.
n.d.=not determined.
[b]
[c]
[d]
methyl-2-cyclohexanone with an enantiomeric excess NADP+ at a wavelength of 340 nm in the presence of the
substrate (e340 =6.3 mmolÀ1 cmÀ1). A cuvette (1 mL) was
of >99% ee (entry 4).
filled with 960 mL of a buffered solution of a substrate
The acyclic Baylis–Hillman product 7 was also re-
(2 mM; phosphate buffer: pH 7.0, 50 mM), and 20 mL of a
duced by means of the At5b-StR from A. thaliana, al-
buffered solution of NADPH (NADPH: 12.5 mM; phos-
though in this preliminary experiment conversion was
phate buffer: pH 7.0, 50 mM). A solution (20 mL) of At5b-
low with 15% (Table 1, entry 5).
StR from A. thaliana (purified; NADPH-dependent; 2.7 mg/
In conclusion, our studies revealed that, besides ste-
mL) was then added. The relative activities were deter-
mined by comparison of measured enzyme activities for the
substrates (in U/mL) with the one for progesterone (re-
garded as the reference experiment with a relative activity
roids as substrates, D4,5-steroid 5b-reductases are also
capable of reducing small molecules bearing an acti-
vated C=C double bond such as monocyclic enones
and acyclic enoate esters. As preferred steroid reduc- of 100%).
tase for this purpose, At5b-StR from A. thaliana was
used. In part, higher enzyme activities have been
found than the one for the steroid substrate progester-
one. Successful preliminary biotransformations with
enzymatic in situ cofactor recycling confirmed the
synthetic potential of steroid reductases for small
molecules with an activated C=C-double bond. Cur-
rently, investigations of scope and limitations of these
types of reduction processes with At5b-StR from A.
Protocol for Preparative Biotransformations
(According to Table 1)
In a 100-mL round-bottom flask, a buffered solution of D4,5-
steroid 5b-reductase from A. thaliana (At5b-StR; HEPES-
KOH buffer, 5% glycerol; pH 8; prepared according to
ref.[11]; entries 1 and 2: 0.945 mL, 3.29 mg/mL; entry 4:
1.15 mL, 4.20 mg/mL; entry 5: 1.12 mL, 2.7mg/mL) or D4,5-
steroid 5b-reductase from D. (I.) canariensis (HEPES-KOH
thaliana are in progress with a focus on the develop- buffer, 5% glycerol; pH 8; prepared according to ref.[13]
;
entry 3: 0.945 mL, 1.22mg/mL) and a glucose dehydrogen-
ase from Bacillus sp. (18 U; Amano Enzymes Inc.) were
subsequently added to a solution of the enone or enoate
ester (2: 0.41 mmol; 4: 0.15 mmol; 7: 0.25 mmol), d-glucose
(2 mmol), and cofactor NADP+ (0.04 mmol) in phosphate
buffer (pH 7; 50 mM; entries 1–4: 70 mL; entry 5: 25 mL).
The reaction mixture was stirred for 24–48 h (for details and
reaction temperature, see Table 1), and subsequently the re-
action mixture was extracted with dichloromethane (en-
tries 1–3: three times 80 mL; entry 4: five times 80 mL;
entry 5: five times 30 mL). The combined organic phases
ment of processes with high volumetric productivities.
Experimental Section
Protocol for the Spectrophotometric Measurement of
Enzyme Activity (According to Scheme 1)
The enzymatic activity was measured spectrophotometrical-
ly as the consumption of NADPH through oxidation to
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Edyta Burda et al.
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were dried over magnesium sulfate, filtered and concentrat-
ed carefully under vacuum. The resulting crude product was
then analyzed by means of proton NMR spectroscopy, and
the conversion was determined from these data by compari-
son with data of reference samples (entries 1–4) or literature
data (entry 5: for literature data, see ref.[15]). The determina-
tion of the enantiomeric excess (entry 4) was carried out by
means of chiral gas chromatography (Shimadzu GC-17 A;
column: CP-Chirasil-DEX; gas carrier: helium; flow:
1.3 mL/min; temperature program: 5 min at 608C, followed
by an increase of 58C per min to 1908C and 10 min at
1908C).
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2008, 73, 4295–4298; e) H. S. Toogood, A. Fryszkow-
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Gardiner, G. M. Stephens, N. S. Scrutton, Adv. Synth.
Catal. 2008, 350, 2789–2803.
[5] Review: S. Buchholz, H. Grçger, in: Biocatalysis in the
Pharmaceutical and Biotechnology Industries, (Ed.:
R. N. Patel), CRC Press, New York, 2006, chapter 32,
pp 757–790.
[6] F. Chamouleau, C. Hagedorn, O. May, H. Grçger, Fla-
vour Fragrance J. 2007, 22, 169–172.
[7] Review: M. Kataoka, M. Kita, M. Wada, Y. Yasohara,
J. Hasegawa, S. Shimizu, Appl. Microbiol. Biotechnol.
2003, 62, 437–445.
Acknowledgements
[8] For one of the rare examples of highly efficient enzy-
matic reduction of an activated C=C double bond with
excellent productivity data, see refs.[2a,2b]
[9] M. Hall, C. Stueckler, H. Ehammer, E. Pointner, G.
Oberdorfer, K. Gruber, B. Hauer, R. Stuermer, W.
Kroutil, P. Macheroux, K. Faber, Adv. Synth. Catal.
2008, 350, 411–418.
The authors thank Ms. Nina Richter for analytical support,
and Evonik Degussa GmbH, Amano Enzymes Inc. and Ori-
ental Yeast Company Ltd. Japan for generous support with
chemicals.
[10] For other recently applied enoate reductases of non-
plant origin, see, for example: a) J. F. Chaparro-Rig-
gers, T. A. Rogers, E. Vazquez-Figueroa, K. M. Polizzi,
A. S. Bommarius, Adv. Synth. Catal. 2007, 349, 1521–
1531; b) ref.[9]
[11] V. Herl, G. Fischer, V. A. Reva, M. Stiebritz, Y. A.
Muller, F. Mꢀller-Uri, W. Kreis, Biochimie 2009, 91,
517–525.
[12] A. Thorn, C. Egerer-Sieber, C. M. Jꢂger, V. Herl, F.
Mꢀller-Uri, W. Kreis, Y. A. Muller, J. Biol. Chem. 2008,
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[13] V. Herl, G. Fischer, R. Bçtsch, F. Mꢀller-Uri, W. Kreis,
Planta Med. 2006, 72, 1163–1165.
[14] Another enzyme which accepts a small enone (2-cyclo-
hexenone) besides steroids (e.g., prednisone) is the
pentaerythritol tetranitrate reductase: T. M. Barna, H.
Khan, N. C. Bruce, I. Barsukov, N. S. Scrutton, P. C. E.
Moody, J. Mol. Biol. 2001, 310, 433–447.
[15] M. Qiu, D.-Y. Wang, X.-P. Hu, J.-D. Huang, S.-B. Yu, J.
Deng, Z.-C. Duan, Z. Zheng, Tetrahedron: Asymmetry
2009, 20, 210–213.
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