Enzymatic ketone reduction: mapping the substrate profile of a short-chain alcohol dehydrogenase (YMR226c) from Saccharomyces cerevisiae

Article abstract of DOI:10.1016/j.tetasy.2007.08.008

A short-chain alcohol dehydrogenase (YMR226c) from Saccharomyces cerevisiae was cloned and expressed in Escherichia coli, and the encoded protein was purified. The activity and enantioselectivity of this recombinant enzyme were evaluated with a series of ketones. The alcohol dehydrogenase (YMR226c) was found to effectively catalyze the enantioselective reductions of aryl-substituted acetophenones, α-chloroacetophenones, aliphatic ketones, and α- and β-ketoesters. While the enantioselectivity for the reduction of β-ketoesters was moderate, the acetophenone derivatives, aromatic α-ketoesters, some substituted α-chloroacetophenones, and aliphatic ketones were reduced to the corresponding chiral alcohols with excellent enantioselectivity. The enantiopreference of this enzyme generally followed Prelog's rule for the simple ketones. The ester functionality played some role in determining the enzyme's enantiopreference for the reduction of α- and β-ketoesters. The present study serves as a valuable guidance for the future applications of this versatile biocatalyst.

Full text of DOI:10.1016/j.tetasy.2007.08.008

Tetrahedron: Asymmetry 18 (2007) 1799–1803
Enzymatic ketone reduction: mapping the substrate profile
of a short-chain alcohol dehydrogenase (YMR226c)
from Saccharomyces cerevisiae
Yan Yang, Dunming Zhu,* Timothy J. Piegat and Ling Hua*
Department of Chemistry, Southern Methodist University, Dallas, TX 75275, USA
Received 30 May 2007; accepted 2 August 2007
Abstract—A short-chain alcohol dehydrogenase (YMR226c) from Saccharomyces cerevisiae was cloned and expressed in Escherichia coli,
and the encoded protein was purified. The activity and enantioselectivity of this recombinant enzyme were evaluated with a series of
ketones. The alcohol dehydrogenase (YMR226c) was found to effectively catalyze the enantioselective reductions of aryl-substituted aceto-
phenones, a-chloroacetophenones, aliphatic ketones, and a- and b-ketoesters. While the enantioselectivity for the reduction of b-keto-
esters was moderate, the acetophenone derivatives, aromatic a-ketoesters, some substituted a-chloroacetophenones, and aliphatic
ketones were reduced to the corresponding chiral alcohols with excellent enantioselectivity. The enantiopreference of this enzyme gen-
erally followed Prelog’s rule for the simple ketones. The ester functionality played some role in determining the enzyme’s enantioprefer-
ence for the reduction of a- and b-ketoesters. The present study serves as a valuable guidance for the future applications of this versatile
biocatalyst.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
selectivity of enzymes is very valuable and instructive in guid-
ing the selection of appropriate biocatalysts for a specific
transformation. Furthermore, correlating substrate profiles
of the alcohol dehydrogenases with their sequential and
structural information will provide valuable insight into
our understanding of how enzymes control activity and
enantioselectivity, and thus facilitate our efforts to
(semi)rationally design novel enzymes with desired sub-
strate specificity and enantioselectivity. With this perspec-
tive in mind, we have studied substrate specificity and
enantioselectivity for the carbonyl reductases from red
yeast Sporobolomyces salmonicolor AKU 4429 and Candida
magnoliae with various ketones of diverse structures, and
have found that these carbonyl reductases catalyze reduc-
tions of aryl, alkyl ketones, and a- and b-ketoesters to
the corresponding chiral alcohols with excellent enantio-
meric purity.18–20 Stewart et al. also systematically investi-
gated the catalytic properties of 18 alcohol dehydrogenases
from baker’s yeast Saccharomyces cerevisiae with a- and
b-ketoesters as the substrates, and these studies provided
valuable information for the future application of these
enzymes.21 However, one interesting alcohol dehydro-
genase encoded by gene YMR226c, which belongs to the
short-chain alcohol dehydrogenase family,22 is not covered
in their elegant studies. Recently, it has been reported that
Chiral alcohols are important and valuable intermediates
in the synthesis of pharmaceuticals and other fine chemi-
cals. The asymmetric reduction of prochiral ketones is
one of the straightforward approaches used to access enan-
tiomerically pure alcohols. In this regard, a variety of chiral
metal complexes have been developed as catalysts for
asymmetric ketone reductions.1–4 An alternative to the
chemical asymmetric reduction process is biocatalytic
transformation using isolated enzymes or whole-cell micro-
organisms, which offers advantages such as mild and envi-
ronmentally benign reaction conditions, high chemo-,
regio-, and stereoselectivity, and is void of residual metals
in the products.5–7 Therefore, biocatalytic ketone reduction
has attracted the attention of both academic and industrial
communities, and many examples with isolated alcohol
dehydrogenases have been described.8–17 However, the sub-
strate profile evaluation of these enzymes has usually been
limited to one or two categories of ketones, although the
information about the substrate specificity and enantio-
*
Corresponding authors. Tel.: +1 214 768 1609; fax: +1 214 768 4809
(D.Z.); e-mail addresses: dzhu@smu.edu; lhua@smu.edu
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.08.008

1800
Y. Yang et al. / Tetrahedron: Asymmetry 18 (2007) 1799–1803
bioreduction of bicyclo[2.2.2]octane-2,5-dione and bicyclo-
[2.2.2]oct-7-ene-2,5-dione by genetically engineered yeast,
which over-expressed YMR226c gene, resulted in (1R,4R,
5S)-5-hydroxybicyclo[2.2.2]octan-2-one (>99% ee) and
(1S,4S,5S)-5-hydroxybicyclo[2.2.2]octan-2-one (98% ee),
(1R,4R,5S)-5-hydroxybicyclo[2.2.2]oct-7-en-2-one (>99%
ee), and (1S,4S,5S)-5-hydroxybicyclo[2.2.2]oct-7-en-2-one
(93% ee), respectively.23,24 This indicates that the alcohol
dehydrogenase encoded by gene YMR226c may have great
potential in the synthesis of chiral building blocks. There-
fore, the gene YMR226c was cloned and expressed in
E. coli by following a modified literature procedure.22
The recombinant protein was purified and assayed against
a variety of ketones. Herein, we present the activity and
enantioselectivity of the recombinant alcohol dehydroge-
nase YMR226c toward the reduction of aryl-substituted
acetophenones, a-chloroacetophenones, aliphatic ketones,
and a- and b-ketoesters, which will serve as a guideline
for the future application of this enzyme in the synthesis
of useful chiral intermediates.
a NADPH regeneration system consisting of D-glucose
dehydrogenase (GDH) and D-glucose (Scheme 1).25 The
enantiomeric excess (ee) values of the product alcohols
were determined by chiral GC or HPLC analysis. The
absolute configurations of product alcohols were assigned
by comparing the retention time with
a
standard
sample.18,19
O
R
R'
NADPH
NADP+
D-Gluconic acid
GDH
D-Glucose
YMR226c
OH
R
R'
Scheme 1. Ketone reduction catalyzed by a yeast alcohol dehydrogenase
YMR226c using a NADPH regeneration system consisting of D-glucose
dehydrogenase (GDH) and D-glucose.
As shown in Table 1, the yeast alcohol dehydrogenase
YMR226c catalyzed the reduction of various b-ketoesters.
Activities were not greatly affected by the structures of R
groups. Compared with the other S. cerevisiae alcohol
dehydrogenases, enzyme YMR226c shows lower enantio-
selectivity for the reduction of aliphatic b-ketoesters.21
The R groups exerted a relatively significant effect on
enantioselectivity, which decreased as the chain length of
R groups became longer. This alkyl chain dependence
has not been observed for the other yeast alcohol dehydro-
genases,21 and might be due to the longer alkyl chain desta-
bilizing the lower-energy substrate orientation in the active
site, leading to the formation of an L-enantiomer (Fig. 2,
A). When the R group was changed to iso-propyl, D-enan-
tiomer was obtained in 87% ee, indicating that the iso-pro-
pyl group further destabilized conformation A in Figure 2
and that orientation B was preferred. A trifluoromethyl
group also inverted the lower-energy substrate conforma-
tion in the enzyme active site as the D-enantiomer was
the major product. The similar alkyl-induced reversal of
enantiopreference has also been observed for three other
2. Results and discussion
An open reading frame YMR226c of the yeast genome
encoded an enzyme, which is a member of the short-chain
alcohol dehydrogenase family. This gene was amplified
from the genomic DNA of S. cerevisiae, and the amplified
DNA fragment was cloned and expressed in E. coli. The
encoded protein was purified and lyophilized as a white
powder, and the SDS-polyacrylamide gel electrophoresis
is shown in Figure 1. The enzyme could be stored at 4 ꢁC
for months without significant loss of activity.
Table 1. Reduction of b-ketoesters catalyzed by yeast alcohol dehydro-
genase YMR226c
O
O
OH
O
OH O
YMR226c
+
R
OEt
R
OEt
R
OEt
Figure 1. The SDS-polyacrylamide gel electrophoresis of the purified
D
L
enzyme. Left lane, protein marker; right lane, purified protein.
R
Specific activitya
eeb (%)
CH3
C2H5
n-C3H7
iso-C3H7
CH2Cl
CF3
26
27
23
23
73
19
45
84 (L)
38 (L)
16 (L)
87 (D)
43 (L)
58 (D)
85 (L)
The recombinant alcohol dehydrogenase was evaluated
toward the reductions of various ketones with diverse
structures. The specific activity of the enzyme was deter-
mined spectrophotometrically by measuring the oxidation
of NADPH at 340 nm at room temperature. Activity assay
with the control cell-free extract, which was obtained by
expression of pTxB1 vector without YMR226c gene in
E. coli Rosetta2(DE3) strain, did not show any activity to-
ward all the substrates tested. The enantioselectivity of the
enzyme toward the reduction of ketones was studied using
C6H5
a The specific activity was defined as nmol of NADPH oxidized in 1 min by
1 mg of enzyme (nmmol minꢀ1 mgꢀ1).
b The enantiomeric excess (ee) values of the product alcohols were deter-
mined by chiral GC analysis.

Y. Yang et al. / Tetrahedron: Asymmetry 18 (2007) 1799–1803
1801
shown in Figure 1, and ethyl (S)-2-phenyl-2-hydroxyace-
tate was obtained in enantiomerically pure form. It was
interesting to note that the reductions of aromatic a-keto-
esters with substituents on the phenyl ring produced (R)-
configured a-hydroxyesters as the major products in excel-
lent enantiomeric purity, implying that the substituents on
the phenyl ring inverted the substrate conformation in the
enzyme active site from A to B. This might be due to the
bulkiness of the substituted phenyl ring, which failed to
be accommodated into the hydrophobic binding pocket
of the active site. For ethyl 3,3-dimethyl-2-oxo-butyrate
and ethyl 3-methyl-2-oxo-butyrate, (R)-enantiomeric a-
hydroxyesters were the major products. This indicated that
substrate conformation B was preferred for these a-keto-
esters and it was consistent with the observation for ethyl
4-methyl-3-oxo-pentanionate.
NMN
NMN
Hδ-
C
Hδ-
C
EtO2C
R
Hδ+
Hδ+
O
O
R
EtO2C
B
A
H
C
H
C
EtO2C
R
OH
OH
R
CO2Et
L-alcohol
D-alcohol
Figure 2. A catalytic site model for alcohol dehydrogenase.
The yeast alcohol dehydrogenase YMR226c also effectively
catalyzed the reductions of aryl-substituted acetophenone
derivatives. The results are presented in Table 3. From
the table it can be seen that the enzyme activity was depen-
dent on the substituents on the benzene ring of acetophe-
none derivatives, although no apparent trend was
observed. The position of the substituent affected the
enzyme activity. For example, the activity decreased in
the order of 40-chloroacetophenone > 30-chloroacetophe-
none > 20-chloroacetophenone. For the methyl substituent,
the meta-substituted substrate shows higher activity than
the para-substituted counterpart. For all the aryl ketones
tested, the product alcohols were obtained in excellent
enantiomeric excess and the enantiomeric preference
followed Prelog’s rule.
alcohol dehydrogenases from baker’s yeast S. cerevisiae.21
However, when the R group was a planar benzene ring,
the reduction gave an L-enantiomer with 85% ee.
The reductions of aromatic and aliphatic a-ketoesters cat-
alyzed by this enzyme were also examined and the results
are summarized in Table 2. The substituents on the phenyl
ring exerted some effect on enzyme activity, and it seemed
that the electron-withdrawing substituents increased the
activity while the electron-donating groups demolished
the activity. Compared with the unsubstituted ethyl 2-
phenyl-2-oxo-acetate, the activity for substrates with F, Cl,
Br, and CN substituents increased, while the substrate with
a methyl group shows lower activity. For aliphatic a-keto-
esters, this enzyme was surprisingly more active toward
the reduction of ethyl 3,3-dimethyl-2-oxo-butyrate than
less sterically crowded ethyl 3-methyl-2-oxo-butyrate.
Table 3. Reduction of aryl ketones catalyzed by the yeast alcohol
dehydrogenase YMR226c
O
OH
Table 2. Reduction of a-ketoesters catalyzed by the yeast alcohol
dehydrogenase YMR226c
YMR226c
R
R
O
OH
CO2Et
OH
R CO2Et
YMR226c
+
R
Specific activitya
eeb (%)
R
CO2Et
R
40-H
20
504
78
31
36
54
19
100
90
99 (S)
99 (S)
99 (S)
99 (S)
98 (S)
99 (S)
99 (S)
99 (S)
98 (S)
40-Cl
R
S
40-Br
40-CH3
40-CF3
R
Specific activitya
eeb (%)
C6H5
40-FC6H4
101
173
263
193
70
472
126
25
99 (S)
94 (R)
99 (R)
98 (R)
99 (R)
91 (R)
98 (R)
28 (R)c
59 (R)c
40-CH3O
20-Cl
30-Cl
30-CH3
40-ClC6H4
40-BrC6H4
40-CH3C6H4
40-CNC6H4
30,50-F2C6H3
iso-C3H7
a The specific activity was defined as nmol of NADPH oxidized in 1 min by
1 mg of enzyme (nmmol minꢀ1 mgꢀ1).
b The enantiomeric excess (ee) values of the product alcohols were deter-
mined by chiral GC analysis.
tert-C4H9
130
a The specific activity was defined as nmol of NADPH oxidized in 1 min by
1 mg of enzyme (nmmol minꢀ1 mgꢀ1).
b The enantiomeric excess (ee) values of the product alcohols were deter-
mined by chiral HPLC analysis unless indicated otherwise.
c The ee values were measured by chiral GC analysis.
Optically active chlorohydrins are versatile intermediates
for the syntheses of biologically active compounds of
pharmaceutical and agricultural interest. For example,
(R)-2-chloro-1-(40-fluorophenyl)ethanol has been used as
a building block in the preparation of optically pure N-
(p-fluorophenyl)hydroxyethyl-4-(p-fluorophenyl)hydroxy-
Similar to ethyl 3-phenyl-3-oxo-propionate, ethyl 2-phenyl-
2-oxo-acetate adopted orientation A in the active site as

1802
Y. Yang et al. / Tetrahedron: Asymmetry 18 (2007) 1799–1803
methyl-piperidine, a novel nonnarcotic analgesics.26 Re-
cently, Chung et al. have used optically active 2-chloro-1-
(20,40-difluorophenyl)ethanol to construct (3S,4R)-1-tert-
butyl-4-(2,4-difluorophenyl)pyrrolidine-3-carboxylic acid,
a drug candidate for treatment of melanocortin receptor
mediated diseases such as obesity and diabetes.27,28 There-
fore, the enzymatic reductions of a series of a-chloroaceto-
phenone derivatives were investigated with the yeast
alcohol dehydrogenase YMR226c. The specific activity
and ee values are presented in Table 4. The results show
that the substituent on the phenyl ring exerted effects on
enzyme activity and enantioselectivity. In all cases, (R)-
configured chlorohydrins were obtained, which followed
the Prelog’s rule as the chlorine changed the CIP priority
although CH2Cl is smaller than phenyl group.
be seen that the chain length did not significantly affect
the enzyme activity. In all cases, (S)-configured alcohols
were produced as the major products, as predicted by Pre-
log’s rule. The methyl ketones were reduced with high
enantioselectivity (>84% ee), while the enantioselectivity
for the reduction of 3-octanone was moderate.
3. Conclusion
The alcohol dehydrogenase (YMR226c) from S. cerevisiae
has been cloned and the encoded protein has been purified.
The substrate profile (activity and enantioselectivity) of this
recombinant enzyme has been evaluated with various
structurally diverse ketones. The alcohol dehydrogenase
(YMR226c) shows a broad substrate preference and effec-
tively catalyzes enantioselective reductions of aryl-substi-
tuted acetophenones, a-chloroacetophenone derivatives,
aliphatic ketones, and a- and b-ketoesters. Especially, the
acetophenone derivatives, aromatic a-ketoesters, and some
of a-chloroacetophenone derivatives and aliphatic ketones
are reduced to the corresponding chiral alcohols with excel-
lent enantioselectivity. The enantiopreference of this
enzyme generally follows Prelog’s rule for the aliphatic
and aromatic ketones, while both the ester functionality
and steric factor are important in determining the enzyme’s
enantiopreference for the reduction of a- and b-ketoesters.
This may be due to the possible hydrogen bonding of ester
group with the enzyme. Therefore, the alcohol dehydro-
genase (YMR226c) is a versatile biocatalyst for asymmetric
reductions of a variety of ketones, and the present study
will provide valuable guidance for further application of
this enzyme in the synthesis of important pharmaceutical
and agricultural intermediates.
Table 4. Reduction of a-chloroacetophenone derivatives catalyzed by the
yeast alcohol dehydrogenase YMR226c
O
OH
Cl
Cl
YMR226c
R
R
R
Specific activitya
eeb (%)
40-H
40-F
40-Cl
75
68
48
35
26
31
15
36
40
18
98 (R)
99 (R)c
99 (R)
43 (R)
56 (R)
99 (R)
96 (R)c
99 (R)
84 (R)
97 (R)
40-NO2
40-CH3CONH
30-NO2–40-CH3CONH
20,40-F2
30-Cl
30,40-Cl2
30,40-(OH)2
a The specific activity was defined as nmol of NADPH oxidized in 1 min by
1 mg of enzyme (nmmol minꢀ1 mgꢀ1).
b The enantiomeric excess (ee) values of the product alcohols were deter-
mined by chiral HPLC analysis unless indicated otherwise.
c The ee values were measured by chiral GC analysis.
4. Experimental
4.1. General remarks
Chiral HPLC analysis was performed on an Agilent 1100
series high-performance liquid chromatography system
with (S,S)-Whelk-O 1 column (25 cm · 4.6 mm, Regis
Technologies Inc.). Chiral GC analysis was performed on
a Hewlett Packard 5890 series II plus gas chromatograph
equipped with autosampler, EPC, split/splitless injector,
FID detector, and CP-Chirasil-Dex CB chiral capillary
column (25 m · 0.25 mm). Ketones and alcohol standard
samples were purchased from Sigma–Aldrich or prepared
as previously reported.19,29
Asymmetric reduction of simple aliphatic ketones is usually
more of a challenge for both chemical and biocatalytic
approaches. The yeast alcohol dehydrogenase YMR226c
was also examined with several aliphatic ketones as the
substrates. The results are summarized in Table 5. It can
Table 5. Reduction of aliphatic ketones catalyzed by the yeast alcohol
dehydrogenase YMR226c
O
OH
R'
YMR226c
4.2. Gene expression and purification of YMR226c
R
R'
R
R
R0
Specific activitya
eeb (%)
The alcohol dehydrogenase gene YMR226c was cloned
from S. cerevisiae by following the literature procedure.22
The gene YMR226c was cloned into pTXB1 expression
vector at the Nde I/BamH I restriction sites to yield plas-
mid pYY23.0, which was transformed into E. coli Roset-
ta2(DE3) strain for expression. Overnight culture was
diluted into fresh LB medium containing ampicillin
(100 lg/mL) and chloramphenicol (34 lg/mL), and incu-
bated at 37 ꢁC until the optical density reached 1.0 at
CH3
CH3
CH3
C2H5
n-C5H11
n-C6H13
n-C7H15
n-C5H11
14
16
20
17
98 (S)
98 (S)
84 (S)
48 (S)
a The specific activity was defined as nmol of NADPH oxidized in 1 min by
1 mg of enzyme (nmmol minꢀ1 mgꢀ1).
b The enantiomeric excess (ee) values of the product alcohols were deter-
mined by chiral GC analysis.

Y. Yang et al. / Tetrahedron: Asymmetry 18 (2007) 1799–1803
1803
595 nm. The expression was induced by 0.1 mM IPTG and
References
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mixed with an equal volume of PEI solution (0.25% poly-
ethyleneimine, NW 40–60K, 6% NaCl, 100 mM Borax,
pH 7.4) to remove lipids. The supernatant was precipitated
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solution was desalted by gel filtration into potassium phos-
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ethanol). Lyophilization of the enzyme solution afforded
the YMR226c enzyme as a white powder.
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We thank Southern Methodist University for financial sup-
port, and this work is also partially supported by Labora-
tory of Biocatalysis and Bioprocessing, State Key
Laboratory of Bioreactor Engineering, East China Univer-
sity of Science and Technology, Shanghai, China.