Asymmetric ketone reduction by a hyperthermophilic alcohol dehydrogenase. The substrate specificity, enantioselectivity and tolerance of organic solvents

Article abstract of DOI:10.1016/j.tetasy.2006.10.042

In an effort to search for effective biocatalysts for asymmetric ketone reduction, the substrate specificity and enantioselectivity of an alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus have been evaluated. This hyperthermophilic alcohol dehydrogenase catalyzes the reduction of various ketones including aryl ketones, α- and β-ketoesters. Interestingly, aryl ketones, phenyl-substituted α- and β-ketoesters were reduced to the corresponding chiral alcohols in an enantiomerically pure form, while the substrates lacking phenyl groups were reduced with a moderate enantioselectivity. It thus suggests that a phenyl group next to the carbonyl group could be very helpful for achieving an excellent enantioselectivity, and this could provide valuable guidance for the future application of this useful enzyme through rational substrate engineering. The reaction temperature increased the enzyme activity, but exerted no effect on the enantioselectivity. This alcohol dehydrogenase also showed a high tolerance of organic solvents such as dimethyl sulfoxide, iso-propanol, methyl tert-butyl ether, and hexane, a particularly important and useful feature for the reduction of ketones with a low solubility in aqueous buffers.

Full text of DOI:10.1016/j.tetasy.2006.10.042

Tetrahedron: Asymmetry 17 (2006) 3010–3014
Asymmetric ketone reduction by a hyperthermophilic
alcohol dehydrogenase. The substrate specificity,
enantioselectivity and tolerance of organic solvents
Dunming Zhu, Hiba T. Malik and Ling Hua*
Department of Chemistry, Southern Methodist University, Dallas, TX 75275, USA
Received 13 October 2006; accepted 30 October 2006
Available online 28 November 2006
Abstract—In an effort to search for effective biocatalysts for asymmetric ketone reduction, the substrate specificity and enantioselectivity
of an alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus have been evaluated. This hyperthermophilic
alcohol dehydrogenase catalyzes the reduction of various ketones including aryl ketones, a- and b-ketoesters. Interestingly, aryl ketones,
phenyl-substituted a- and b-ketoesters were reduced to the corresponding chiral alcohols in an enantiomerically pure form, while
the substrates lacking phenyl groups were reduced with a moderate enantioselectivity. It thus suggests that a phenyl group next to
the carbonyl group could be very helpful for achieving an excellent enantioselectivity, and this could provide valuable guidance for
the future application of this useful enzyme through rational substrate engineering. The reaction temperature increased the enzyme activ-
ity, but exerted no effect on the enantioselectivity. This alcohol dehydrogenase also showed a high tolerance of organic solvents such as
dimethyl sulfoxide, iso-propanol, methyl tert-butyl ether, and hexane, a particularly important and useful feature for the reduction of
ketones with a low solubility in aqueous buffers.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Enzymes from hyperthermophiles, that is, microorganisms
that grow optimally above 80 ꢁC, usually display not only
an extreme stability at a high temperature and high pres-
sure, but also a high tolerance of chemical denaturants
such as organic solvent.19 In addition, the hyperthermo-
philic enzymes can be subjected to heat treatment, which
is an easy handling and effective protein purification
process. These unique features are desired for certain
applications. Therefore, hyperthermophilic enzymes are
interesting from both a scientific point of view and indus-
trial applications.20,21 Recently, Machielsen et al. have
cloned and biochemically characterized an alcohol dehydro-
genase from the hyperthermophilic archaeon Pyrococcus
furiosus.22 This enzyme shows a high resistance to thermal
inactivation, which has a half-life of 130 min at 100 ꢁC. In
addition, this alcohol dehydrogenase shows a preference
for the less expensive co-factor NADH, although it takes
both NADH and NADPH as a co-factor. These unique
properties make this enzyme a potential biocatalyst for
practical use and stimulated us to further assess its sub-
strate specificity, stereoselectivity, and tolerance of organic
solvents. Herein we report our findings on this unique alco-
hol dehydrogenase, which would provide useful guidance
for the future application of this enzyme.
Asymmetric reduction of ketones is a very important trans-
formation in organic synthesis because chiral carbinols are
useful bioactive compounds and their precursors. Among
the methodologies available, asymmetric biocatalytic
reduction has attracted a great attention from the green
chemistry perspective.1–5 Thus, there is a constant demand
for efficient biocatalysts for this vital transformation, and
many new biocatalytic systems both in whole cell and iso-
lated enzyme forms have recently been developed.6–12 As
part of our own effort to develop an effective ketoreductase
tool-box of isolated enzymes, we are especially interested in
ketoreductases which complement the currently available
ones.13–15 More specifically, we have particularly been
searching for ketoreductases with the following properties,
such as anti-Prelog enantioselectivity,16 broader substrate
specificity,17 larger active site for bulky substrates,17,18
higher thermostability18 and tolerance of organic solvents.
*
Corresponding author. Tel.: +1 214 768 1609; fax: +1 214 768 4089;
e-mail: lhua@smu.edu
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.10.042

D. Zhu et al. / Tetrahedron: Asymmetry 17 (2006) 3010–3014
Table 2. Reduction of a-ketoesters catalyzed by PFADH
3011
2. Results and discussion
O
OH
OH
The alcohol dehydrogenase gene adhD from the hyper-
thermophilic archaeon P. furiosus was cloned and
expressed in Escherichia coli and the recombinant enzyme
was purified by heat treatment, and lyophilized (see Section
4). The obtained alcohol dehydrogenase (PFADH) was
stable for months at 4 ꢁC. The enzyme activity was assayed
toward various ketones including aryl ketones and a- and
b-ketoesters by spectrophotometrically measuring the
oxidation of NADH at 340 nm. The enantioselectivity for
the reduction of various ketones catalyzed by PFADH
was evaluated using NADH as a co-factor, which was
regenerated with a recycling system comprising glucose
dehydrogenase and glucose (Scheme 1). The results are
presented in Tables 1–3.
PFADH
+
R
R
R
CO2Et
CO2Et
CO2Et
ee (%)
R
Relative activitya
C6H5
754
631
915
1092
523
1469
646
769
946
654
99 (R)
99 (R)
99 (R)
99 (R)
99 (R)
99 (R)
99 (R)
99 (R)
44 (S)
71 (S)
40-F–C6H4
40-Cl–C6H4
40-Br–C6H4
40-CH3–C6H4
40-CN–C6H4
30,50-F2–C6H4
30,40-Cl2–C6H4
iso-C3H7
tert-C4H9
a The specific activity of acetophenone was 13 nmol minꢀ1 mgꢀ1 at 37 ꢁC
and its relative activity was defined as 100.
R
R'
NADH
NAD+
D-Gluconic acid
GDH
D-Glucose
O
PFADH
R R'
Table 3. Reduction of b-ketoesters catalyzed by PFADH
R
R'
+
O
O
OH
O
OH
O
PFADH
OH
OH
+
R
OEt
R
OEt
R
OEt
ee (%)
Scheme 1. Reduction of various ketones catalyzed by PFADH with
NADH regeneration system.
R
Relative activitya
CH3
C2H5
C3H7
iso-C3H7
CH2Cl
C6H5
285
162
215
108
1092
154
95 (S)
60 (S)
24 (R)
16 (S)
4 (R)
From Table 1, it can be seen that the alcohol dehydro-
genase from the hyperthermophilic archaeon Pyrococcus
furiosus catalyzed the reduction of acetophenone deriva-
tives to the corresponding (S)-chiral alcohols in an enantio-
merically pure form. The substituents on the benzene ring
of the aryl ketones exerted some effect on the enzyme activ-
ity, although the influence was not dramatic. The enantio-
selectivity of the reduction was not affected by the
substituents and pattern of the substitution. The a-chlori-
nated acetophenone showed a much higher activity than
the unsubstituted one (more than 10 times).
99 (S)
a The specific activity of acetophenone was 13 nmol minꢀ1 mgꢀ1 at 37 ꢁC
and its relative activity was defined as 100.
Table 2 summarizes the results of the PFADH-catalyzed
reduction of aromatic and aliphatic a-ketoesters at 37 ꢁC.
It can be seen that PFADH was more active toward the
reduction of a-ketoesters than the above discussed aryl
ketones. The enzyme activity was affected by the substituents
on the benzene ring of aromatic a-ketoesters. For example,
the enzyme activity increased when the para-substituents
were F, Cl, and Br. The aromatic a-ketoester with para-
CN substituent showed the highest activity. For aliphatic
a-ketoesters, the enzyme activity decreased when iso-propyl
was replaced with tert-butyl, suggesting that the bulkiness
of the R group might inhibit the enzyme activity. It is inter-
esting to note that the alcohol dehydrogenase from P. furio-
sus catalyzed the reduction of aromatic a-ketoesters to the
corresponding enantiomerically pure (R)-configured a-
hydroxyesters, while the enantioselectivity of the aliphatic
a-ketoester reduction was moderate with the major pro-
ducts being (S)-enantiomer. Similar enantiopreference
reversals have previously been observed in the reduction
of a-ketoesters catalyzed by a carbonyl reductase from
Sporobolomyces salmonicolor AKU4429 (SSCR) and
enzyme–substrate docking studies showed that the opposite
orientations of aromatic and aliphatic a-ketoesters in the
active cavity of SSCR enzyme was responsible for the enan-
tiopreference flip-flop.17 Aromatic and aliphatic a-keto-
esters may also take opposite orientations in the active
Table 1. Reduction of aryl ketones catalyzed by PFADH
O
OH
X
X
PFADH
R
R
X
R
Relative activitya
ee (%)
H
H
H
H
H
H
H
H
H
H
H
Cl
40-H
100
162
131
77
108
146
231
285
154
157
131
1331
99 (S)
99 (S)
99 (S)
99 (S)
99 (S)
99 (S)
99 (S)
99 (S)
99 (S)
99 (S)
99 (S)
99 (R)
40-F
40-Cl
40-Br
40-CH3
40-CF3
20-Cl
30-Cl
30-CH3
20-OCH3
30,50-(CF3)2
40-H
a The specific activity of acetophenone was 13 nmol minꢀ1 mgꢀ1 at 37 ꢁC
and its relative activity was defined as 100.

3012
D. Zhu et al. / Tetrahedron: Asymmetry 17 (2006) 3010–3014
site of the PFADH enzyme, thus the different enantiomers
were obtained as the major products. The excellent enantio-
selectivity for aromatic a-ketoesters also suggested that
the phenyl group next to the carbonyl functionality might
play a critical role in the substrate–protein interaction to
distinguish the faces at which the hydride transfer occurred.
solubility of hydrophobic ketones.24 However, the presence
of organic solvents deactivates the enzymes in many cases.
Enzymes, which are tolerant of organic solvents, will thus
be of great interest from both scientific and practical points
of view. Therefore, the reductions of ethyl 2-(4-cyanophen-
yl)-2-oxo-acetate in various organic solvents were exam-
ined to assess the organic solvent tolerance of the alcohol
dehydrogenase from the hyperthermophilic archaeon P.
furiosus. The reductions were carried out under two condi-
tions: (a) 2 equiv of NADH and (b) a catalytic amount of
NADH with a regeneration system consisting of D-glucose
dehydrogenase and D-glucose. The reaction mixtures were
incubated at 30 ꢁC for 16 h. The products and the recov-
ered substrates were extracted into methyl tert-butyl ether.
The conversion and ee values were determined by chiral
HPLC analysis. The conversions are presented in Figure
2. The results showed that the alcohol dehydrogenase from
the hyperthermophilic archaeon P. furiosus was active in
reaction media, with up to 30% of water-miscible solvents,
such as DMSO and iso-propanol. The low conversion with
NADH recycling system in the buffer–DMSO mixture (3 in
Fig. 2) should be due to the inactivation of D-glucose
dehydrogenase, because 100% conversion was obtained
with a stoichiometric amount of NADH. This hyperthermo-
philic alcohol dehydrogenase was also active in two phase
systems containing methyl tert-butyl ether or hexane, but
the presence of toluene, ethyl acetate or n-butyl acetate
inactivated this enzyme. It was worth noting that an excel-
lent enantioselectivity of 99% ee was observed in all the
cases. This indicated that the alcohol dehydrogenase from
the hyperthermophilic archaeon P. furiosus was a very
robust enzyme in some organic solvents. From a synthetic
point of view, this property was particularly important and
useful for the reduction of ketones with a low solubility in
aqueous buffers.
The PFADH-catalyzed reduction of several b-ketoesters
was also studied (Table 3). Ethyl 3-oxo-butyrate was
reduced to ethyl (S)-3-hydroxybutyrate in a high enantio-
meric purity. As the size of the R group increased, the
enantioselectivity of the reductions decreased, with the
exception of R being a phenyl group, in which the product
ethyl (S)-3-hydroxy-2-phenylpropionate was obtained in
99% ee. Although the size of R groups did not significantly
affect the activity, an electron-withdrawing group such as Cl
greatly enhanced the enzyme activity. This is similar to the
above observation for a-chloroacetophenone (Table 1).
Since the alcohol dehydrogenase from the hyperthermo-
philic archaeon P. furiosus showed high resistance to ther-
mal inactivation,22 it would be interesting to study the
temperature effects on the reactivity and enantioselectivity
of this enzyme. Therefore, ethyl 2-(4-cyanophenyl)-2-oxo-
acetate was used as the substrate for the reduction at differ-
ent temperatures. The reductions were performed under
two conditions, that is, (a) 2 equiv of NADH and (b) a
catalytic amount of NADH with a regeneration system
consisting of D-glucose dehydrogenase and D-glucose. The
reactions were quenched after 1 h. The reaction mixtures
were extracted with methyl t-butyl ether. The conversion
and ee values were then determined by HPLC analysis.
The conversions are presented in Figure 1. It can be seen
from the results that the enzyme activity enhanced as the
reaction temperature increased. Interestingly, ethyl (4-
cyanophenyl)-2-hydroxyacetate was obtained in an essen-
tially enantiomerically pure form at all temperatures. This
is in contrast to the previous observation that the enantio-
selectivity decreases at higher reaction temperatures.23
Stoichiometric amount of NADH
NADH recycling system
100
80
60
40
20
0
Stoichiometric amount of NADH
NADH recycling system
120
100
80
60
40
20
0
1
2
3
4
5
6
7
8
9
10
Solvent
Figure 2. PFADH-catalyzed reduction of ethyl 2-(4-cyanophenyl)-2-oxo-
acetate in various organic solvents. (1) Potassium phosphate buffer; (2)
15% DMSO; (3) 30% DMSO; (4) 15% iso-propanol; (5) 30% iso-propanol;
(6) 30% methyl tert-butyl ether; (7) 30% toluene; (8) 30% ethyl acetate; (9)
30% n-butyl acetate; (10) 30% hexane.
30
40
50
60
70
80
Temperature (°C)
Figure 1. Temperature effect on the enzyme activity of the alcohol
dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus
toward the reduction of ethyl 2-(4-cyanophenyl)-2-oxo-acetate.
3. Conclusion
The substrate specificity and enantioselectivity of an alco-
hol dehydrogenase from the hyperthermophilic archaeon
P. furiosus (PFADH) have been assessed toward a variety
of ketones including aryl ketones, a- and b-ketoesters. This
hyperthermophilic alcohol dehydrogenase catalyzed the
Enzymatic reduction of poorly water-soluble ketones in
aqueous media is usually hampered by the low availability
of substrates. One strategy to overcome this limitation is
the introduction of organic solvents, which improve the

D. Zhu et al. / Tetrahedron: Asymmetry 17 (2006) 3010–3014
3013
asymmetric reduction of these ketones to the correspond-
ing chiral alcohols. The temperature-dependent studies
indicated that the enzyme activity enhanced as the reaction
temperature increased. Surprisingly, the enantioselectivity
did not change at higher temperatures. The latter was not
consistent with the previous observations that the enantio-
selectivity decreased as the reaction temperature
increased.23 This was a useful feature for certain applica-
tions requiring asymmetric reduction at high temperatures.
Another interesting observation was that aryl ketones,
phenyl-substituted a- and b-ketoesters were reduced to
the corresponding chiral alcohols in an enantiomerically
pure form, while the substrates without phenyl groups were
reduced with a moderate enantioselectivity. This suggested
that a phenyl group next to the carbonyl group might be
required to achieve an excellent enantioselectivity. The
chloro group at the a-carbon of the carbonyl group
improved the enzyme activity as shown in the reductions
of a-chloroacetophenone and ethyl 4-chloro-3-oxo-
butyrate. These observations will provide useful guidance
for the future application of this unique enzyme through
rational substrate design. The present alcohol dehydro-
genase also showed a high tolerance of organic solvents,
such as dimethyl sulfoxide, iso-propanol, methyl tert-butyl
ether, and hexane. This feature would be particularly
important and useful for the reduction of hydrophobic
ketones with a low availability in pure aqueous buffers.
DNA containing this alcohol dehydrogenase gene was
transformed into Rosetta2(DE3)pLysS E. coli strain
(Novagen). Overnight pre-cultures were diluted into LB
containing 100 lg/ml of ampicillin and 34 lg/ml of chlor-
amphenicol, the cells were induced with 0.5 mM of IPTG
when optical density at 595 nm was 0.62. The bacterial
cultures were incubated at 30 ꢁC on an orbital shaker at
180 rpm for another 5 h.
The cultures of E. coli Rosetta2(DE3)pLysS were harvested
by centrifugation. The cell pellet was re-suspended in
potassium phosphate buffer (20 mM, pH 7.3), and the cells
were lysed by homogenizer. The cell-free extract was
obtained after being centrifuged at 11,000 rpm (ꢁ15,000g)
for 30 min. The resulting cell-free extract was heated in a
water bath at 80 ꢁC for 30 min, and centrifuged at
4100 rpm for 30 min. The obtained supernatant was con-
centrated and centrifuged at 4100 rpm for 15 min. The
resulting enzyme solution was lyophilized. The lyophilized
PFADH enzyme was confirmed by protein gel analysis and
used for enzymatic reactions.
4.2. Activity assay of the alcohol dehydrogenase
The activity of alcohol dehydrogenase toward the reduc-
tion of ketones (Tables 1–3) was determined by spectro-
photometrically measuring the oxidation of NADH at
340 nm (e = 6.22 mMꢀ1 cmꢀ1) in the presence of excess
ketones. The activity was measured at 37 ꢁC in a 96-well
plate, in which each well contained ketone substrates
(6.25 mM), NADH (0.25 mM) in potassium phosphate
buffer (100 mM, pH 6.5, 180 ll). The reaction was initiated
by the addition of alcohol dehydrogenase PFADH (20 ll
solution containing 20 or 40 lg of enzyme). The specific
activity was defined as the number of nmol of NADH
converted in one minute by 1 mg of enzyme (nmol ·
minꢀ1 · mgꢀ1).
4. Experimental
The chiral HPLC analysis was performed on an Agilent
1100 series high-performance liquid chromatography sys-
tem with (S,S)-Whelk-O 1 column (25 cm · 4.6 mm, Regis
Technologies Inc.). The chiral GC analysis was performed
on a Hewlett–Packard 5890 series II plus gas chromato-
graph equipped with autosampler, EPC, split/splitless
injector, FID detector, and CP-Chirasil-Dex CB chiral
capillary column (25 m · 0.25 mm). The alcohol dehydro-
genase activities toward the reduction of ketones were
assayed using SpectraMax M2 microplate reader (Molecu-
lar Devices). All the ketones and alcohol standard samples
were purchased from Sigma–Aldrich or prepared as
reported previously.17,18
4.3. Enantioselectivity of ketone reduction catalyzed by
alcohol dehydrogenase
The enantioselectivity of the enzymatic reduction of
ketones was studied using an NADH recycling system.
The general procedure was as follows: D-glucose (4 mg),
D-glucose dehydrogenase (0.5 mg), NADH (0.5 mg), alco-
hol dehydrogenase (1 mg) and ketone solution in DMSO
(50 ll, 0.25 M) were mixed in a potassium phosphate buffer
(1 ml, 100 mM, pH 6.5) and the mixture was shaken over-
night at 37 ꢁC. The mixture was extracted with methyl tert-
butyl ether (1 ml). The organic extract was dried over anhy-
drous sodium sulfate and subjected to either chiral HPLC
or GC analysis to determine the enantiomeric excess. The
absolute configurations of product alcohols were identified
by comparing the chiral HPLC or GC data with the stan-
dard samples as reported previously.17,18,25
4.1. Gene expression and purification of alcohol
dehydrogenase
The alcohol dehydrogenase gene adhD from the hyper-
thermophilic archaeon P. furiosus (GenBank Accession
number AE010289) was amplified from genomic DNA
(gift from Frank T. Robb) by forward primer 50-
GCGCGCCATGGCAAAAAGGGTAAATGCATTCAA-
CGA-30 (the Nco I restriction site is underlined) and the
reverse primer 50-TTTTGGGATCCTCACACACACCT-
CCTTGCCATCT-30 (the BamH I restriction site is under-
lined). The amplification was performed in a final volume
of 100 ll, and the reaction mixtures contained 100 ng of
genomic DNA, 50 pmol of each primer, 200 lM of dNTP,
1 · PCR buffer, 1.25 U of Pfx DNA polymeras and 1 mM
MgSO4. The adhD gene was cloned into pET15b expres-
sion vector at the (NcoI/BamH I) sites and the plasmid
4.4. The reduction of ethyl 2-(4-cyanophenyl)-2-oxo-acetate
in various organic solvents
The reductions of ethyl 2-(4-cyanophenyl)-2-oxo-acetate in
various organic solvents were performed as follows: (a)
NADH (14 mg), alcohol dehydrogenase (1 mg) and ketone

3014
D. Zhu et al. / Tetrahedron: Asymmetry 17 (2006) 3010–3014
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solution in DMSO (40 ll, 0.25 M) were mixed in a mixture
(1 ml) of potassium phosphate buffer (100 mM, pH 6.5)
and appropriate amount of solvent; (b) D-glucose (4 mg),
D-glucose dehydrogenase (0.5 mg), NADH (0.5 mg), alco-
hol dehydrogenase (1 mg) and ketone solution in DMSO
(40 ll, 0.25 M) were mixed in a mixture (1 ml) of potassium
phosphate buffer (100 mM, pH 6.5) and appropriate
amount of solvent. The mixture was shaken at 30 ꢁC for
16 h. The mixture was extracted with methyl tert-butyl
ether (1 ml). The organic extract was dried over anhydrous
sodium sulfate and subjected to chiral HPLC analysis to
determine the conversion and enantiomeric excess.
4.5. The reduction of ethyl 2-(4-cyanophenyl)-2-oxo-acetate
at different temperatures
The reductions of ethyl 2-(4-cyanophenyl)-2-oxo-acetate at
37, 45, 60, and 75 ꢁC were performed as follows: (a)
NADH (14 mg), alcohol dehydrogenase (0.5 mg) and
ketone solution in DMSO (40 ll, 0.25 M) were mixed in
a mixture (1 ml) of potassium phosphate buffer (100 mM,
pH 6.5); (b) D-glucose (4 mg), D-glucose dehydrogenase
(0.5 mg), NADH (0.5 mg), alcohol dehydrogenase
(0.5 mg), and the ketone solution in DMSO (40 ll,
0.25 M) were mixed in a mixture (1 ml) of potassium phos-
phate buffer (100 mM, pH 6.5). The mixture was shaken at
the specified temperature for 1 h. The mixture was
extracted with methyl tert-butyl ether (1 ml). The organic
extract was dried over anhydrous sodium sulfate and was
subjected to chiral HPLC analysis to determine the conver-
sion and enantiomeric excess.
15. Zhu, D.; Mukherjee, C.; Hua, L. Tetrahedron: Asymmetry
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Acknowledgements
We thank Frank T. Robb from University of Maryland
Biotechnology Institute for providing the genomic DNA
of the hyperthermophilic archaeon Pyrococcus furiosus
and Southern Methodist University for generous financial
support.
21. de Miguel Bouzas, T.; Barros-Velazquez, J.; Villa, T. G.
Protein Pept. Lett. 2006, 13, 645–651.
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