Asymmetric synthesis of (S)-ethyl-4-chloro-3-hydroxy butanoate using a Saccharomyces cerevisiae reductase: Enantioselectivity and enzyme-substrate docking studies

Article abstract of DOI:10.1016/j.bbapap.2010.06.011

Ethyl (S)-4-chloro-3-hydroxy butanoate (ECHB) is a building block for the synthesis of hypercholesterolemia drugs. In this study, various microbial reductases have been cloned and expressed in Escherichia coli. Their reductase activities toward ethyl-4-chloro oxobutanoate (ECOB) have been assayed. Amidst them, Baker's yeast YDL124W, YOR120W, and YOL151W reductases showed high activities. YDL124W produced (S)-ECHB exclusively, whereas YOR120W and YOL151W made (R)-form alcohol. The homology models and docking models with ECOB and NADPH elucidated their substrate specificities and enantioselectivities. A glucose dehydrogenase-coupling reaction was used as NADPH recycling system to perform continuously the reduction reaction. Recombinant E. coli cell co-expressing YDL124W and Bacillus subtilis glucose dehydrogenase produced (S)-ECHB exclusively.

Full text of DOI:10.1016/j.bbapap.2010.06.011

Biochimica et Biophysica Acta 1804 (2010) 1841–1849
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Asymmetric synthesis of (S)-ethyl-4-chloro-3-hydroxy butanoate using a
Saccharomyces cerevisiae reductase: Enantioselectivity and enzyme–substrate
docking studies
Jihye Jung a, Hyun Joo Park a, Ki-Nam Uhm b, Dooil Kim c, Hyung-Kwoun Kim a,
⁎
a
Division of Biotechnology, The Catholic University of Korea, Bucheon 420-743, Republic of Korea
Equispharm Ltd., Daejeon 305-811, Republic of Korea
b
c
Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Ethyl (S)-4-chloro-3-hydroxy butanoate (ECHB) is a building block for the synthesis of hypercholesterolemia
drugs. In this study, various microbial reductases have been cloned and expressed in Escherichia coli. Their
reductase activities toward ethyl-4-chloro oxobutanoate (ECOB) have been assayed. Amidst them, Baker's
yeast YDL124W, YOR120W, and YOL151W reductases showed high activities. YDL124W produced (S)-ECHB
exclusively, whereas YOR120W and YOL151W made (R)-form alcohol. The homology models and docking
models with ECOB and NADPH elucidated their substrate specificities and enantioselectivities. A glucose
dehydrogenase-coupling reaction was used as NADPH recycling system to perform continuously the
reduction reaction. Recombinant E. coli cell co-expressing YDL124W and Bacillus subtilis glucose
dehydrogenase produced (S)-ECHB exclusively.
Received 2 February 2010
Received in revised form 5 June 2010
Accepted 14 June 2010
Available online 20 June 2010
Keywords:
Reductase
Enantioselectivity
Chiral intermediate
Hyperlipidemia drug
Docking model
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Kamitori et al. previously elucidated the X-ray crystal structures of
Sporobolomyces salmonicolor carbonyl reductase and its complex with
The asymmetric reduction of ketones to optically pure alcohols has
become the focus of a great deal of attention, primarily in regard to their
possible use as chemotherapeutic drugs and chiral building blocks. Many
oxidoreductase enzymes from a variety of microorganisms are capable of
reducing carbonyl groups with chemo-, regio-, and stereoselectivity [1,2].
Enzymatic ketone reductions can be achieved via the use of either
isolated enzymes [1,3,4] or whole cell systems [5–7]. The use of isolated
enzymes is advantageous in that undesirable enantiomer formation
mediated by contaminating ketoreductases is minimized, and the
enzymes can be stored and employed as normal chemical reagents. On
the other hand, whole cell biocatalysts are more stable, due to the
protective effects of the cell membrane. Enzyme purification is
unnecessary when whole cells are applied as biocatalysts. Additionally,
the reductase reaction can be performed using endogenous NAD(P)H as
a cofactor, even without the addition of external NAD(P)H. If
recombinant Escherichia coli cells co-expressing both a specific
reductase and glucose dehydrogenase (GDH) are used, not only is
undesirable enantiomer formation minimized, but the continuous
recycling of NAD(P)H is also enabled [5,6,8].
NADPH (PDB ID 1Y1P) [9]. They were the first to propose the
hydrogen bonding residues that fix the carbonyl oxygen of substrates
in the active site and hydrophobic channel. Hua et al. conducted a
molecular modeling analysis of different aryl alkyl ketones in the 1Y1P
active site in order to gain insight into the enantioselectivity observed
in the aryl alkyl ketone reduction [10].
In a previous study, we generated 10 different microbial
reductases [11]. In this work, we screened 3 enzymes among them,
which were capable of generating each enantiomer of ethyl 4-chloro-
3-hydroxy butanoate (ECHB), a chiral intermediate in hyperlipidemia
drugs. According to the results of the product analysis and the protein
structure modeling study, YDL124W was finally selected for the
production of (S)-ECHB. The properties of the selected carbonyl
reductase were characterized and recombinant E. coli cells performing
the reductase-glucose dehydrogenase-coupling reaction were con-
structed in order to generate (S)-ECHB efficiently.
2. Materials and methods
2.1. Chemicals
Abbreviations: ECOB, ethyl-4-chloro-3-oxo butanoate; ECHB, ethyl-4-chloro-3-
hydroxy butanoate; GDH, glucose dehydrogenase; NAD(P)H, nicotinamide adenine
dinucleotide (phosphate); DMSO, dimethyl sulfoxide; GC, gas chromatography
Ethyl-4-chloro-3-oxo butanoate (ECOB), glucose dehydrogenase
(GDH, Thermoplasma acidophilum), NAD(P)H, and NAD(P)+ were
purchased from Sigma-Aldrich Co. (St. Louis, MO). (R)- and (S)-ethyl-
⁎
Corresponding author. Tel.: +82 2 2164 4890; fax: +82 2 2164 4865.
E-mail address: hkkim@catholic.ac.kr (H.-K. Kim).
1570-9639/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2010.06.011

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J. Jung et al. / Biochimica et Biophysica Acta 1804 (2010) 1841–1849
4-chloro-3-hydroxy butanoate ((R)- and (S)-ECHB) were received
from Equispharm Ltd. (Korea). All other chemicals were of analytical
grade.
of their energy (DOPE) and cubic spline restraints integrated in
MODELLER. The side chains were then modeled while avoiding steric
clashes, but also keeping the existing other side chains automatically.
The model structure is then optimized by the method of conjugate
gradients combined with molecular dynamics and simulated anneal-
ing [24]. This method was used to relieve steric clashes and overlaps of
side chains. The loop refinement relies on an atomistic distance-
dependent statistical potential of mean force for non-bond interaction
[25]. The stereochemical properties of minimized structures were
evaluated by Ramacandran plot calculations computed with Procheck
program [26]. Profile-3D [27,28] further confirms the environment
of side chains and examines the misfolded regions. RMSD fit was
calculated by alignment of structural homology feature in MODELLER.
2.2. Reductase and glucose dehydrogenase activity assay
Reductase activity was evaluated at 30 °C by measuring the
reduction in absorbance at 340 nm for 5–10 min using a spectropho-
tometer. The reaction mixture (1 mL) consisted of 1 mM ECOB
(100 mM stock in DMSO), 0.2 mM NAD(P)H, 50 mM potassium
phosphate (pH 6.5) buffer, and 5–50 μL of cell-free extract or 10 μL of
the permeabilized cells. One unit of enzyme was defined as the quantity
of enzyme required to catalyze the oxidation of 1 μmol NAD(P)H in
1 min at 30 °C.
The oxidation reaction mixture (1 mL) of glucose dehydrogenase
consisted of 10 mM glucose, 0.2 mM NADP+, 50 mM potassium
phosphate (pH 6.5) buffer, and 0.1 μL of commercial glucose
dehydrogenase or 10 μL of the permeabilized cells. The reaction rate
was monitored using a spectrophotometer on the basis of the increase
in absorbance at 340 nm for 5–10 min at 30 °C. One unit of enzyme
was defined as the quantity required to reduce 1 μmol of NADP+ in
1 min at 30 °C.
2.4. Purification of reductase YDL124W
Reductase YDL124W was identified as an appropriate enzyme for
the reduction of ECOB to (S)-ECHB in an enantioselective fashion. The
reductase protein in the cell-free extract was purified as follows. First,
the cell-free extract was loaded onto a Ni-NTA column (QIAGEN
GmbH, Hilden) equilibrated with a 50 mM Tris–HCl buffer (pH 7.8)
containing 50 mM imidazole and 300 mM NaCl. After washing with
the same buffer, the YDL124W was eluted from the column via the
application of a 200 mM imidazole buffer. The active fractions were
then collected and desalted with a PD-10 desalting column (GE
Healthcare Bio-Sciences AB, UK).
2.3. Computational methods
The 3D model structure of YOL151W was determined in previous
studies [11]. In this research, the initial 3D model structures of
YDL124W and YOR120W from Sacharomyces cerevisiae were con-
structed on the basis of the crystal structures of two aldo-keto
reductases (PDB code: 3BUV [12] and 1C9W [13], respectively). The
identity of the entire amino acid sequences of YDL124W and 3BUV
was calculated to be 30.7% by ClustalW method in DNASTAR program.
The identity of YOR120W and 1C9W was calculated to be 39.4% by the
same method.
The target structures were modeled in silico via a structural
homology search using HHpred/HHsearch [14,15] and homology
modeling using MODELLER [16], then optimized using FoldX [17]. The
active sites of YDL124W and YOR120W were evaluated via compar-
ison with two corresponding X-ray templates. The geometries of the
substrate (ECOB) and cofactor (NADPH) were optimized using the
Hartree-Fock method with a 6-31G* basis set as implemented in the
Gaussian 03 program (Gaussian, Inc., Wallingford, CT). The restrained
electrostatic potential procedure of the Antechamber module from
the AMBER suite was used to generate input files with charges for
docking programs [18,19]. The results were then utilized as valid
inputs for the AutoDock ligand preparation procedure.
Docking was conducted with AutoDock (ver. 4.0), using the
implemented empirical free energy function and the Lamarckian
genetic algorithm [20]. The best docked conformations were those
determined to have the lowest binding energy and the greatest
number of members in the cluster, thereby indicating good conver-
gence. The best orientation was identified and optimized using the
scoring function based on the AMBER force field FF99 and energy
minimization according to the Nelder and Mead algorithm [19] for
induced-fit simulation. The parameters embedded in the AMBER
package were used for energy minimization and molecular dynamics
[21,22]. Molecular dynamics (MD) simulations were also conducted.
Additionally, the docking of ECOB and NADPH to our target proteins
was initially assessed in accordance with the topological binding sites
predicted by several algorithms [23].
Structurally variable region (loop, Ile120-Lys127) was generated
by sampling main-chain dihedral angels built randomly using the
discrete optimized protein energy (DOPE)-based loop modeling
protocol in MODELLER software. Each loop takes the initial conforma-
tion and randomizes it by 3 Å in each of the Cartesian coordinates.
Thousand loop structures for Ile120-Lys127 were ranked on the basis
2.5. Effect of temperature and pH
The effects of temperature and pH were evaluated using the
purified YDL124W enzyme. Reactions were monitored spectrophoto-
metrically by measuring the reduction in NADPH at 340 nm with
the purified enzyme. The reaction rate was measured at various
temperatures (5–60 °C). Meanwhile, in order to evaluate temperature
stability, the enzyme was pre-incubated for 30 min at 10–45 °C, after
which the remaining activity was assayed at 30 °C. The following
buffers (50 mM) were utilized to assess the effects of pH: pH 3–6.5,
acetic acid/sodium acetate; pH 6.5–8, KH2PO4/K2HPO4; pH 7.5–9,
Tris–HCl; pH 9–10.5, glycine–KCl–KOH. To confirm the pH stability,
the enzyme was pre-incubated in the pH buffers listed above for
30 min on ice, then adjusted to pH 6.5, under which conditions the
enzyme's residual activity was evaluated.
2.6. Effect of concentration of buffer and DMSO
The effect of the potassium phosphate buffer concentration on the
reductase reaction rate was measured. Various concentrations (50,
100, 150, 200, 250, 300 mM) of potassium phosphate buffer (pH 6.5),
including 1 mM ECOB, 0.2 mM NADPH and 5 μL of purified enzyme
were used.
Reaction solutions (50 mM potassium phosphate buffer, pH 6.5)
containing 1 mM ECOB, 0.2 mM NADPH, 5 μL of purified enzyme,
and various concentrations of DMSO (final conc. 1, 3, 10, 15, 20, and
30%, v/v) were mixed, and the reaction rates were measured by
determining the decrease in absorbance at 340 nm.
2.7. Enzymatic coupling reaction and reaction mixture analysis
The enantioselectivity of the enzymatic reduction of ECOB was
evaluated. The general procedure was as follows: 100 mM ECOB,
1 mM NADPH, 13 units of reductase YDL124W, 150 mM D-glucose,
and 26 units of commercial glucose dehydrogenase were mixed in a
total volume of 15 mL of potassium phosphate buffer (50 mM, pH
6.5), and the mixture was incubated at 25 °C. The pH of the reaction
mixture was monitored with a pH meter and maintained at 6.0–6.5
via the addition of 1 M NaOH.

J. Jung et al. / Biochimica et Biophysica Acta 1804 (2010) 1841–1849
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For the coupling reactions using the whole cell systems, 100 mM
ECOB, 1 mM NADPH, 150 mM D-glucose, and 13 units (reductase
activity) of the permeabilized cells containing two plasmids (pETR124
and pACGDH) or one plasmid (pACR124-GDH), were mixed in a total
volume of 15 mL of potassium phosphate buffer (50 mM, pH 6.5), and
the mixture was incubated at 25 °C.
Aliquots (100 μL) of the reaction mixture were sampled, mixed
with 400 μL of ethyl acetate and 50 μL of saturated K2CO3, then
centrifuged for 10 min at 10000×g. The ethyl acetate (250 μL) layer
was obtained, dried via speedvac, dissolved with methylene chloride
(400 μL), and mixed with 20 μL of pyridine and 10 μL of acetyl chloride.
After 1 h of incubation, the mixture was washed with 1 M HCl (200 μL)
and saturated NaHCO3 (200 μL). The organic phase was then analyzed
using a GC system equipped with a chirasil-dex column (Varian LTD.).
The column temperature was increased from 70 °C to 180 °C at a rate of
5 °C per min and maintained for 2 min at 180 °C. A frame ionization
detector was used to analyze the quantity of alcohols generated by
enzymatic reduction. By comparing the retention times and peak areas
of standard (R)- and (S)-alcohol peaks, the total quantities (in moles)
of products in the reaction mixtures were calculated.
HindIII and then ligated downstream of the first T7 promoter of the
pACYGDH vector and used to transform E. coli BL21 (DE3). The
recombinant plasmid was designated as pACR124-GDH.
E. coli cells containing one vector (pACR124-GDH) or two vectors
(pETR124 plus pACGDH) were cultured at 20° C in Luria–Bertani
medium (1% tryptone, 0.5% yeast extract and 0.5% NaCl) containing
100 μg/mL of ampicillin (for pETR124) and 170 μg/mL of chloram-
phenicol (for pACR124-GDH and pACGDH). When the cells reached an
optical density (OD600 nm) of 0.5, expression was induced via the
addition of 1 mM isopropyl thio-β-D-galactoside (IPTG). Cultivation
was continued for an additional 24 h, after which the cells were
harvested via 10 min of centrifugation at 10,000×g at 4° C and
suspended in a 1/20 volume of 50 mM potassium phosphate buffer
(pH 6.5).
To render the cell membrane permeable to the substrates and
cofactor, the recombinant cells were mixed and shaken at 300 rpm
and 30° C for 10 min with 5 mM EDTA and 1% toluene [29], washed by
10 min of centrifugation at 3,000×g, and used for the whole cell
coupling reaction.
3. Results
2.8. Co-expression of reductase and glucose dehydrogenase in E. coli
3.1. Screening of enantioselective reductase
To obtain Bacillus subtilis glucose dehydrogenase, a PCR primer set
was designed on the basis of the nucleotide sequence information in
the NCBI GenBank database (EF626962.1) as follows: 5’-CGCCATATG-
TATCCGGATTTAAAAGGA-3’/5’-ATAGGTACCTTAACCGCGGCCTGCCTG-
3’. The genomic DNA of B. subtilis was used as a template. The PCR
conditions were as follows: 30 cycles of 95° C for 1 min, 43° C for
1 min and 72° C for 1 min. The PCR product was cloned into pGEM-T
(Promega Corp., Madison, WI) and transformed into E. coli XL1-Blue.
The Bacillus GDH gene in pGEM-T vector was cut with NdeI and KpnI,
then ligated downstream of the second T7 promoter of the
pACYCDuet-1 vector and used to transform E. coli BL21 (DE3). The
recombinant plasmid was designated as pACGDH.
A recombinant plasmid harboring both the YDL124W gene and the
Bacillus GDH gene was also constructed as follows. In order to subclone
the YDL124W reductase gene into pACGDH, the following PCR primer
set was designed: 5’-GAGCCATGGCATCATTTCACCAACAGTTC-3’/5’-
GCGAAGCTTTTATACTTTTTGAGCAGC-3’. The recombinant plasmid
pET-YDL124 constructed in the previous research [11] was used as a
template. The PCR conditions were identical to those described above.
The PCR product was cloned into pGEM-T and transformed into E. coli
XL1-Blue. The YDL124W gene in pGEM-T vector was cut with NcoI and
In a previous study, we generated 10 different microbial
reductases in E. coli cells [11]. First, their reductase activities toward
ECOB were measured via a spectrophotometric method (Table 1).
Reductase activity was assayed in accordance with the basic principle
that NAD(P)H was oxidized when the substrate was reduced by the
enzyme. The majority of the E. coli cell-free extracts evidenced
measurable reductase activity (0.1–6.6 U/mL) toward ECOB (Table 1).
Among them, the cell-free extracts containing YDL124W, YOR120W,
and YOL151W evidenced relatively high levels of activity. GC analysis
was subsequently performed to detect ECHB enantiomers as the
reaction products. YDL124W reductase exclusively generated (S)-
ECHB, but YOR120W and YOL151W exclusively generated (R)-ECHB
(Table 1).
3.2. Homology modeling and evaluation
To rationalize the observed enantioselectivity of YDL124W,
YOR120W, and YOL151W toward ECOB, we first conducted computer
modeling experiments. It has already been established that the
hydride transfer from NADPH to substrate was the decisive factor in
Table 1
Screening of reductase suitable for the reduction of ECOB to ECHB.
No.
GenBank locus
tag
Reported/putative function
Reductase activity (U/mL)
ΔNAD(P)Ha
ΔECHBb
Saccharomyces cerevisiae
1
2
3
4
5
6
7
8
YJR096W
YDL124W
YOR120W
YGL185C
YPL113C
YDR541C
YCR107W
YOL151W
Aldo-keto reductase
0.161 (NADH)
2.683 (NADPH)
1.108 (NADPH)
0.106 (NADH)
0.093 (NADH)
0.062 (NADH)
0.186 (NADH)
6.658 (NADPH)
—
Alpha-keto amide reductase
Glycerol dehydrogenase
Hydroxy acid dehydrogenase
Glyoxylate reductase
Dihydrokaempferol 4-reductase
Aryl-alcohol dehydrogenase
Methylglyoxal reductase
0.227 (S)
0.243 (R)
—
—
—
—
0.194 (R)
Leuconostoc citreum
9
LCK_01181
NCgl2041
Oxidoreductase
Oxidoreductase
0.199 (NADH)
0.126 (NADH)
—
—
Corynebacterium glutamicum
10
a
Reaction mixtures contain 1 mM ECOB, 0.2 mM NAD(P)H, and 5–50 μL cell-free extract in 1 mL of 50 mM potassium phosphate (pH 6.5). Spectrophotometric assays were used.
Reaction mixtures contain 5 mM ECOB, 1 mM NAD(P)H, 1 U crude reductase (spectrophotometer assay), 7.5 mM glucose, 2 U commercial GDH in 5 mL of 50 mM potassium
b
phosphate (pH 6.5). The reaction products were analyzed using GC method. One unit of enzyme was defined as the amount of enzyme catalyzing the production of 1 μmol ECHB in
1 min at 30 °C.

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J. Jung et al. / Biochimica et Biophysica Acta 1804 (2010) 1841–1849
determining the enantioselectivity of dihydrofolate reductase [30–
32]. We also hypothesized that the enantioselectivity originated in the
hydride transfer step, and thus we focused our computer modeling on
this step.
The 3D model structure of YOL151W was reported recently by our
group [11]. In this study, from a similar structure search by HHpred/
HHsearch [14,15] for YDL124W and YOR120W, two X-ray crystal
structures, 3BUV [12] and 1C9W [13] are obtained as potential
homology templates, respectively. We superimposed the active sites
of two target proteins with each of the X-ray crystal templates. 3D
structural alignments for YDL124W-3BUV and YOR120W-1C9W were
conducted using their entire lengths and secondary structures
(supplementary data 1 and 2).
Initial structures were refined by energy minimization and MD
simulations. Obviously, the total energies of the two systems
remained in equilibrium after 150 ps. The average structures
calculated during the entire 500 ps MD simulations were used as
the final models for YDL124W and YOR120W. The RMS values for the
main chains for YDL124W-3BUV and YOR120W-1C9W were calcu-
lated as 0.72 Å and 1.63 Å, respectively.
Final models of YDL124W and YOR120W were evaluated by
Profile-3D [27,28] and Procheck [26]. The Profile-3D evaluations
yielded compatibility scores for YDL124W and YOR120W of 202 and
199, respectively, which is close to 208, the expected score for a
protein of this size. In the Procheck assessment, the reliability
backbone torsion angles ψ-φ of the target proteins were examined,
and the templates were evaluated for comparison. The percentages of
ψ-φ angles in the core Ramachandran region were 87.1 and 86.3% for
YDL124W and YOR120W, respectively, which are comparable to the
template avg. 89.7%.
YDL124W is slightly greater than that of YOL151W (−44.59 kcal/mol
vs. −40.87 kcal/mol) arising from the more hydrogen bonds. In
addition, representative plots for possible backbone torsion angles of
NADPH in complex with YOL151W were presented in supplementary
data 5 and 6.
In the ECOB-YOR120W complex, our docking results showed that
Tyr56 (corresponding to Tyr48 in 1C9W) and His112 (His110 in
1C9W) may perform important functions in ECOB binding. Tyr56 and
His112 form hydrogen bonds with carbonyl oxygen atoms at the
methyl chloride end of ECOB. Total interaction energy in the complex
is −42.71 kcal/mol. In addition, the aliphatic chain of Leu128 and
aromatic rings of Trp113, which are located around the ethyl group of
ECOB, appears to be relevant to hydrophobic interaction (supple-
lementary data 7).
3.4. Purification and characterization of YDL124W
In an effort to evaluate the reaction properties of YDL124W, we
purified the recombinant enzyme. Because the enzyme harbored a His
tag at its C-terminal region, it could be readily purified via two
sequential purification steps: Ni-NTA column and PD-10 column
chromatography. The purified enzyme had a specific activity of
1.22 U/mg toward the ECOB substrate.
Its optimal reaction conditions were evaluated via the spectro-
photometric method. Analysis of the initial reaction rate showed that
its optimum temperature was 45 °C, and its enzyme activity was
rapidly reduced at temperatures over 45 °C (Fig. 2A). It was stable up
to 25 °C for 30 min, and its thermal stability was rapidly reduced over
30 °C (Fig. 2B). Its optimum pH was 6.5, and it was stable for 30 min at
a pH range of 5–8 (Fig. 2C and D).
In order to convert large quantities of substrate using the
reductase, the reaction process should be coupled with a GDH enzyme
that regenerates NADPH. However, as these coupling reactions
proceeded, a large quantity of glucose was oxidized into gluconic
acid and the pH of the solution was decreased. Therefore, the selection
of a buffer system is a matter of critical importance. YDL124W
reductase activity was measured in an increasing molar concentration
of potassium phosphate buffer (pH 6.5). Reductase activity was
maintained at a constant level in a range of 50–300 mM (Fig. 2E).
On the other hand, as low substrate solubility is another problem
in the enzyme reaction, the substrate was dissolved in DMSO to a
concentration of 1 M and then utilized for the reaction. Reductase
activity was measured with an increasing concentration of DMSO in
order to elucidate the effects of DMSO (Fig. 2F). DMSO reduced
enzyme activity only slightly up to a concentration of 3%, but its
inhibitory effects evidenced a detectable increase at concentrations
over 5%.
3.3. Docking of the ECOB into YDL124W, YOL151W and YOR120W
The resulting conformations of ECOB-YDL124W, ECOB-YOL151W,
and ECOB-YOR120W complexes from the docking simulation using
AutoDock 4.0 are shown in Fig. 1A–C, respectively. Interaction
energies between each amino acid of the active site and ECOB were
conducted, with the objective of evaluating the docking results in
general and identifying the significant binding-site residues. The
channel structures of YDL124W, YOL151W, and YOR120W for the
passage of the substrates were presented in supplementary data 3.
Tyr64 and His122 were shown to cast a large shadow on ECOB
bound to YDL124W. They corresponded to Tyr58 and Glu120 in 3BUV.
The carbonyl oxygen connected to the methyl chloride moiety of
ECOB forms hydrogen bonds with Tyr64 and His122. A hydrogen bond
is also formed (avg. distance; 3.39 Å) between the second carbonyl
group of ECOB and Lys30 (supplementary data 4). The latter hydrogen
bond was not found in the x-ray structure of ARK1D1 complex with
steroids. Thus this is one of unique features of ECOB of YDL124W.
These hydrogen bonding interactions may serve to enhance the
stability of the ECOB-YDL124W complex.
The kinetic parameters, kcat and Km values, for ECOB were
calculated as 112 min−1 and 1.08 mM, respectively (Table 2). The
kinetic parameters for NADPH were 102 min−1 and 4.36 μM,
respectively (supplementary data 8).
Ser127 and Tyr165 have been identified as the most important
residues for ECOB binding into the active site of YOL151W. In
YOL151W, these residues are conserved in the 1Y1P, corresponding to
Ser133 and Tyr177, respectively. The carbonyl oxygen connected to
the methyl chloride group of ECOB forms two hydrogen bonds with
Ser127 and Tyr165. Phe87, Phe132, Met134, and Leu235 in the active
site are indispensable residues, and account for the hydrophobic
interactions with ECOB. The total interaction energy of ECOB with
3.5. Production of (S)-ECHB by reductase YDL124W-GDH-coupling
reaction system
To maintain the ketone reduction reaction for a prolonged period,
abundant NADPH must be supplied. However, as NADPH is a rather
expensive material, a NADPH regeneration system is clearly required. In
this study, we utilized a GDH-coupling reaction for NADPH recycling.
Reaction products from the reductase YDL124W-GDH-coupling
reaction were analyzed with a GC system equipped with a chiral
column. (R)-ECHB could not be detected at all, and this result implied
that the enantiomeric excess was almost 100%.
Table 2
Kinetic parameters of YDL124W.
kcat (min−1
)
Km (mM)
kcat/Km (min−1 mM−1
)
As the coupling reaction continued, gluconic acid accumulated and
the pH of the solution was consequently reduced. The optimum pH of
YDL124W reductase was pH 6.5, and its activity rapidly decreased
ECOB
NADPH
112
102
1.08
0.00436
104
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J. Jung et al. / Biochimica et Biophysica Acta 1804 (2010) 1841–1849
1845
Fig. 1. Homology models and docking models. Homology models of YDL124W and YOR120W were constructed based on the crystal structures of 3BUV and 1C9W, respectively. The
homology model of YOL151W was cited [11]. ECOB and NADPH were docked into the enzyme active sites of YDL124W (A), YOL151W (B), and YOR120W (C), respectively. The
coloring scheme shows nitrogen atoms in blue, oxygen atoms in red, carbon atoms in light grey, sulfur atom in yellow, phosphorus atoms in orange, and chlorine atoms in green.
Dotted lines indicate the expected hydrogen bonds.
below pH 6.0. In this experiment, the pH of the solution was maintained
within pH 6.0–6.5 via occasional additions of 1 M NaOH solution.
When the coupling reaction was conducted with initial substrate
concentrations of 100 mM, approximately 70% of the ECOB was
converted to (S)-ECHB with enantiomeric excess of 96% within
240 min (Fig. 3).
plasmids in this research. The pACGDH plasmid contained the
B. subtilis GDH gene (GenBank ID EF626962.1) and the pACR124-
GDH plasmid harbored both the GDH gene and the reductase gene
(Fig. 4A). The results of SDS-PAGE analysis revealed that E. coli cells
containing both pETR124 and pACGDH generated both reductase and
GDH (Fig. 4B) and E. coli cells containing only pACR124-GDH also
generated these two enzymes (Fig. 4B).
3.6. Coupling reaction using an E. coli whole cell system
The reductase and GDH activities of four different recombinant
E. coli cells were measured using a whole cell system (Table 3). As
anticipated, E. coli intact cells containing pETR124 or pACGDH
evidenced either reductase or GDH activities exclusively. E. coli cells
We used the pETR124 plasmid [11] for the production of the
reductase enzyme. Additionally, we constructed two recombinant

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J. Jung et al. / Biochimica et Biophysica Acta 1804 (2010) 1841–1849
Fig. 2. Reaction properties of YDL124W reductase. Reductase activity was determined by measurements of the reduction in NADPH absorption at 340 nm. (A) Reductase activity was
measured at various temperatures. (B) After 30 min of pre-incubation at various temperatures, residual reductase activity was measured at 30 °C. (C) Reductase activity was
measured at various pHs using buffers including acetic acid/sodium acetate (●), potassium phosphate (○), Tris–HCl (■), and glycine–KCl–KOH buffers (□). (D) After 30 min of pre-
incubation at various pHs, residual reductase activity was measured at pH 6.5. (E) The effect of potassium phosphate buffer concentration was determined. (F) The effects of DMSO
concentration were determined. 1% (●), 3% (▲), 10% (■), 15% (□), 20% (◆), 30% (◊).
harboring two plasmids (pETR124 and pACGDH) evidenced both
reductase and GDH activities simultaneously. Additionally, E. coli cells
containing pACR124-GDH exhibited both activities, although the GDH
activity was substantially higher than the reductase activity.
We conducted whole cell coupling reactions using the latter two E.
coli cell systems. The coupling reactions worked well in these E. coli
whole cell systems (Fig. 5). Using a 100 mM initial concentration of
the ECOB substrate, the conversion yield reached a level of 80% within
180 min. (S)-ECHB was exclusively produced with an enantiomeric
excess value of 98% in both reaction systems.
4. Discussion
(S)-ECHB is a chiral intermediate of many hyperlipidemia drugs
including Atorvastatin, as mentioned above. This compound could be
generated from ECOB, a prochiral ketone compound, via the process of
enantioselective reduction. For the efficient production of (S)-ECHB,
therefore, a suitable reductase enzyme should be employed. In this
study, YDL124W, YOL151W, and YOR120W reductases were selected
for the production of chiral ECHB among 10 different microbial
Fig. 3. (S)-ECHB production via the reductase-GDH-coupling reaction. The amounts of
ECOB (■), (S)-ECHB (●), and enantiomeric excess values (○) were measured over the
time course.

J. Jung et al. / Biochimica et Biophysica Acta 1804 (2010) 1841–1849
1847
Fig. 4. Recombinant plasmid construction and SDS-PAGE. (A) Three recombinant plasmids, pETR124 (YDL124W), pACGDH (Bacillus GDH), and pACR124-GDH (YDL124W and GDH),
were constructed. (B) SDS-analysis was conducted for four different recombinant E. coli cells containing pETR124, pACGDH, pETR124 plus pACGDH, and pACR124-GDH, respectively.
RedHis means reductase with His tag. S and P mean supernatant and precipitate after cell lysis via sonication.
reductases. These three enzymes have been reported in several
previous reports, and their substrate specificity and enantioselectivity
toward a variety of compounds have also been characterized. We
confirmed that YDL124W generated (S)-ECHB exclusively, whereas
YOR120W and YOL151W exclusively generated (R)-ECHB.
In this study, in an effort to compare and explain their enantiose-
lective reaction mechanisms at the molecular level, we conducted
homology modeling and docking modeling. Interestingly, three binding
models may be comparable with the substrate binding crystal
structures of 3BUV (corresponding to YDL124W), 1Y1P (YOL151W)
and 1C9W (YOR120W), respectively [9,12,13]. These similarities imply
the reliability of our docking results. The results indicated that three
complexes were similar with regard to their hydrophilic interactions,
but evidenced different enantioselectivity characteristics due to
hydrophobic interactions with surrounding residues.
to catalyze reduction of Δ4-ene double bonds through the 4-pro-R
hydride transferred from the re-face of the nicotinamide ring of the
NADPH cofactor to C5 of the steroid substrate. In contrast, the
substrate in the YDL124W-ECOB complex by automatic AutoDock
docking procedure adopts different anti-conformation as not ob-
served in AKR1D1–steroid complex structure, indicating a different
orientation of the ECOB in the YDL124W. This observation appears to
result from the introduction of the steric bulk of the His120 (YDL124W)
Among three enzymes, we focused on YDL124W reductase
because it exclusively generated the (S)-form product. 3D structure
alignment of binding pocket of AKR1D1–steroid complex with
YDL124W model structure containing bound ECOB is shown Fig. 6.
Δ4-3-ketosteroids are substrates for mammalian steroid carbon-
carbon double bond reductase (ARK1D1), where they mainly undergo
Table 3
Enzyme activity of recombinant E. coli whole cells.
Plasmid
Reductase activity (U/mL)
GDH activity (U/mL)
Fig. 5. Production of (S)-ECHB by whole cell coupling reaction. The conversion yield and
e.e. values were measured over the time course. ECOB (▲) and (S)-ECHB (●) via the one
plasmid (pACR124-GDH) were measured. ECOB (△) and (S)-ECHB (○) via and two-
plasmid systems (pETR124 plus pACGDH,) were also measured. Enantiomeric excess
values by the one-plasmid (■) and two-plasmid systems (□) were measured.
pETR124
pACGDH
pETR124+pACGDH
pACR124GDH
1.95
N.D.
3.35
1.66
N.D.
9.66
3.80
5.29

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J. Jung et al. / Biochimica et Biophysica Acta 1804 (2010) 1841–1849
NADPH cofactor turned upwards shown in Fig. 6, with the carbonyl
group pointing toward the nicotinamide, forming the orientation for a
possible hydride transfer.
YDL124W has already been purified from S. cerevisiae, and its
properties have been characterized [33]. YDL124W has been identified
as an NADPH-dependent alpha-keto amide reductase that exhibits
reducing activity not only for aromatic alpha-keto amides, but also for
aliphatic and aromatic alpha-keto esters. Although the substrate
specificity and stereoselectivity of the enzyme have previously been
reported in the literature, no 3D-structural studies have yet been
conducted. Therefore, an explanation of the reaction mechanism using
the active site–substrate docking model is clearly meaningful.
As shown in our docking model of YDL124W (Figs. 1A and 7A), the
carbonyl oxygen atom of ECOB, which is located close to the
nicotinamide ring, forms hydrogen bonds with Tyr64 and His122.
The C4 atom of the nicotinamide ring may possibly donate its
hydrogen atom to the carbonyl carbon atom of the ECOB, with a
distance of 3.1 Å between the C4 atom and the carbonyl carbon. The
proposed catalytic mechanism is as follows. The carbonyl oxygen
atom of the substrate forms hydrogen bonds with both the Tyr and His
residues, and is protonated from the Tyr residue, followed by the
attack of a hydrogen atom from the C4 atom of NADPH against the
carbonyl carbon atom of the substrate. Meanwhile, the nicotinamide
mononucleotide (NMN) ring of the cofactor is located at the re-face of
ECOB. Thus, this ketone should be reduced to (S)-ECHB, which is
consistent with our experimental observations (Fig. 3). On the other
hand, in the cases of YOR120W and YOL151W, the NMN ring of the
cofactor appeared to be located at the si-face of ECOB. Thus, this
ketone should be reduced to (R)-ECHB (Fig. 7B).
Fig. 6. 3D structure alignment of binding pocket of AKR1D1–steroid complex with
YDL124W model structure containing bound ECOB. NADPH, cortisone, Tyr58, and
Glu120 in the active site of AKR1D1 (magenta color) were compared with NADPH,
ECOB, Tyr64, and His122 in the active site of YDL124W (same colors used in Fig. 1).
imidazole side chain by the replacement of its corresponding Glu120
residue in AKR1D1. As a result, the 4-pro-(R)-hydride of the NADPH
cofactor would be adjacent to the ECOB carbonyl group (to C-5 of the
substrate carbon-carbon double bond in the AKR1D1) in the YDL124W
active site.
The simulations of ECOB bound to the YDL124W were started from
catalytically active pose with the carbonyl group with methyl chloride
of ECOB being positioned close to and oriented toward the
nicotinamide ring of the cofactor in all MD simulations. In the
simulations of the YDL124W-ECOB complex, the 4-pro-(R)-hydride of
Recently, some reductase reactions have been conducted using
whole cell systems. In the current study, we co-expressed both
reductase and Bacillus glucose dehydrogenase in the same E. coli cell,
using one-vector and two-vector systems. The two enzymes were
successfully expressed in the E. coli cells and NADPH recycling was
Fig. 7. Stereochemistry of reductase and schematic representation of the reduction process. (A) YDL124W converted ECOB into (S)-ECHB. (B) YOL151W and YOR120W converted
ECOB into (R)-ECHB.

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1849
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5. Conclusions
Baker's yeast YDL124W, YOR120W, and YOL151W reductases
showed high activities for ECOB. YDL124W produced (S)-ECHB ex-
clusively, whereas YOR120W and YOL151W made (R)-form alcohol.
Homology models and docking models with ECOB and NADPH
elucidated their substrate specificity and enantioselectivity; in the
case of YDL124W, the nicotinamide mononucleotide (NMN) ring of
the cofactor is located at the re-face of ECOB, whereas in the case of
YOR120W and YOL151W, the NMN ring of the cofactor is located at
the si-face of ECOB. Recombinant E. coli cells co-expressing YDL124W
and B. subtilis glucose dehydrogenase could recycle NADPH and con-
vert ECOB into (S)-ECHB continuously.
Acknowledgement
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This work was supported by the 21C Frontier Microbial Genomics
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