Purification and Characterization of an Enone Reductase from Sporidiobolus salmonicolor TPU 2001 Reacting with Large Monocyclic Enones

Article abstract of DOI:10.1002/cctc.201700244

We discovered a novel enone reductase from Sporidiobolus salmonicolor TPU 2001 (SsERD) and expressed the gene in Escherichia coli. The enzyme catalyzed the reduction of (E)-3-methylcyclopentadec-2-en-1-one (E-2), cyclopentadec-2-en-1-one (3), and cyclododec-2-en-1-one (4) to (S)-muscone (S-1), cyclopentadecan-1-one (5), and cyclododecan-1-one (6), respectively. The apparent K_m and V_max values for E-2 were estimated to be 4.9±0.4 μm and 100±1.4 nmol min^?1 mg^?1, respectively. The enzyme was specific to NADPH, and cysteine residues strongly affected the enzyme activity. The enzyme exhibited the highest activity at pH 8.0 and high stability in the pH range from 4.5 to 11.0. Using 10 mU of the enzyme, S-1 was synthesized from 0.1 mm E-2 with 94.8 % yield and 100 % enantiomeric excess by incubation at pH 7.0 and 30 °C for 60 min. We further successfully constructed an enone reductase with high specific activity by mutation of SsERD. The Y67A variant from SsERD exhibited 4.5 times higher specific activity and 3 times higher catalytic efficiency toward E-2. This is the first report of an enzyme catalyzing the reduction of carbon–carbon double bond of large monocyclic enones.

Full text of DOI:10.1002/cctc.201700244

Accepted Article
Title: Purification and characterization of a novel enone reductase
from Sporidiobolus salmonicolor TPU 2001 reacting with large
monocyclic enones
Authors: Kazunori Yamamoto, Yuko Oku, Atsutoshi Ina, Atsushi Izumi,
Masaharu Doya, Syuji Ebata, and Yasuhisa Asano
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ChemCatChem
10.1002/cctc.201700244
FULL PAPER
Purification and characterization of a novel enone reductase from
Sporidiobolus salmonicolor TPU 2001 reacting with large
monocyclic enones
Kazunori Yamamoto,[a,b] Yuko Oku,[a,b] Atsutoshi Ina,[a,b] Atsushi Izumi,[a,b] Masaharu Doya, Syuji
[c]
[
c]
[a,b]
Ebata, and Yasuhisa Asano*
We discovered
a
novel enone reductase from Sporidiobolus
reduction of progesterone to 5-pregnane-3,20-dione.[4]
Morphinone reductase (EC 1.3.1.42) catalyzes reduction of
morphinone to hydromorphone.[5] However, enzymes catalyzing
reduction of large monocyclic enones have not been studied in
detail, although certain chemical methods have been reported.
(R)- or (S)-muscone, (R)- or (S)-3-methylcyclopentadecan-1-
salmonicolor TPU 2001 (SsERD) and expressed the gene in
Escherichia coli. The enzyme catalyzed the reduction of (E)-3-
methylcyclopentadec-2-en-1-one (E-2), cyclopentadec-2-en-1-one
(
3), and cyclododec-2-en-1-one (4) to (S)-muscone (S-1),
cyclopentadecan-1-one (5), and cyclododecan-1-one (6), respectively.
The apparent K and Vmax values for E-2 were estimated to be 4.9 ±
.4 μM and 100  1.4 nmol min mg , respectively. The enzyme was
one,[6] are produced using enantioselective chemical methods
m
-
1
-1
[7,8]
0
(Scheme 1).
(R)-Muscone (R-1), which is well known as a
specific to NADPH, and cysteine residue strongly affected the enzyme
activity. The enzyme exhibited the highest activity at pH 8.0 and high
stability in the pH range from 4.5 to 11.0. Using 10 mU of the enzyme,
S-1 was synthesized from 0.1 mM E-2 with 94.8% yield and 100%
enantiomeric excess by incubation at pH 7.0 and 30 °C for 60 min.
We further successfully constructed enone reductase with high
specific activity by mutation of SsERD. The Y67A variant from SsERD
exhibited 4.5 times higher specific activity and 3 times higher catalytic
efficiency toward E-2. This is the first report of the enzyme catalyzing
reduction of carbon-carbon double bond of large monocyclic enones.
natural perfume, is produced by the enantioselective
hydrogenation of (E)-3-methylcyclopentadec-2-en-1-one (E-2)
using Ru
methylcyclopentadec-2-en-1-one (Z-2) using Ru
binap] NEt (R-Ru-BINAP) with 100% yield and 98%
2
Cl
4
[(S)-p-tolyl-binap]
2
NEt
3
(S-Ru-BINAP) or of (Z)-3-
2
4
Cl [(R)-p-tolyl-
2
3
[
8c]
enantiomeric excess (ee). (S)-Muscone (S-1) is also produced
by the same method from E-2 using R-Ru-BINAP or Z-2 using S-
Ru-BINAP with 100% yield and 98% ee. However, these methods
require expensive Ru-BINAP catalyst and rigorous reaction
conditions.
Introduction
The enzymes catalyzing asymmetric reduction of activated
carbon-carbon double bonds such as α,-unsaturated aldehydes,
ketones, and esters, have been widely studied for application in
production of chiral building blocks in the synthesis of bioactive
compounds.[1] For example, enzymes that react with small ring
enones, such as 5- or 6-membered ring enones, have been
reported. Old yellow enzyme (OYE) (EC 1.6.99.1) catalyzes
reduction of 2- or 3-alkyl-substituted cyclohexen-1-ones to the
corresponding ketones.[2] 12-Oxophytodienoate reductases (EC
1
.3.1.42) are available for asymmetric reduction of 2- and 3-
methylcyclopenten-1-one.[3] Enzymes catalyzing reduction of
carbon-carbon double bond of polycyclic enones have been also
reported. Progesterone 5-reductase (EC 1.3.99.6) catalyzes
Scheme 1. Chemical syntheses of (R)-muscone (R-1) and (S)-muscone (S-1)
by asymmetric hydrogenation using Ru-BINAP catalyst. The structure of Z-2 is
rotated counterclockwise 45 degrees relative to other three compounds.
[
a]
Dr. K. Yamamoto, Y. Oku, Dr. A. Ina, Dr. A. Izumi, Prof. Dr. Y. Asano
Biotechnology Research Center and Department of Biotechnology
Toyama Prefectural University
In this study, we investigated development of biochemical
methods for production of muscone. First, we screened enzymes
catalyzing the reduction of large monocyclic enone E/Z-2, and
isolated the enzyme catalyzing this reduction from Sporidiobolus
salmonicolor TPU 2001. The enzyme responsible for the
reduction of large monocyclic enones and asymmetrically
synthesized S-1, but not R-1. Although S-1 is not useful for
perfumery, the enzymes catalyzing the reduction of large
monocyclic enones are important as catalysts to elucidate the
5
180 Kurokawa, Imizu, Toyama (Japan)
E-mail: asano@pu-toyama.ac.jp
Dr. K. Yamamoto, Y. Oku, Dr. A. Ina, Dr. A. Izumi, Prof. Dr. Y. Asano
Asano Active Enzyme Molecule Project, ERATO, JST
[
b]
c]
5180 Kurokawa, Imizu, Toyama (Japan)
[
Dr. M. Doya, Dr. S. Ebata
Toho Earthtec, Inc.
1450 Kurotori, Nishi-ku, Niigata, Niigata (Japan)
Supporting information for this article is given via a link at the end of
the document.
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ChemCatChem
10.1002/cctc.201700244
FULL PAPER
reaction mechanism of enzymatic reduction of large monocyclic
enones. The present paper describes purification and
characterization of the enzyme from S. salmonicolor TPU 2001.
The optimal conditions for asymmetric synthesis of S-1 are also
described.
Cloning and expression of the SsERD gene, and purification
of recombinant enzyme
It was difficult to obtain a large amount of the enzyme from the
selected strain (only 0.4 mU of enzyme in 1 mL of the culture).
Therefore, we investigated cloning and expression of the SsERD
gene as follows: The SsERD gene was cloned from total cDNA
using primers corresponding to N- and C-terminal amino acid
sequence of CEQ42599, as described in the Experimental section.
The amino acid sequence deduced from the cloned SsERD gene
showed 92% identity to that of CEQ42599. In addition, above
amino acid sequences of the N-terminal and six internal peptides
were contained in the deduced amino acid sequence (Figure S2).
The nucleotide sequence of SsERD was registered in
DDBJ/EMBL/GenBank (accession number: LC209205). The
SsERD gene was next ligated with the pET28a vector, and
Escherichia coli BL21 (DE3) was transformed by the plasmid
harboring the SsERD gene. In this expression experiment, the
strain harboring the expression vector produced 10.2 mU of
enzyme per 1 mL of the culture, indicating that the enzyme
productivity was increased 25 times using the recombinant
method. The recombinant enzyme purified using the same
procedure as that used for the native enzyme (Table S1) exhibited
similar specific activity (101 mU mg-1 of protein) as that of the
native enzyme. Therefore, we used the purified recombinant
enzyme in following studies.
Results
Screening and purification of enone reductase reacting with
large monocyclic enones
In preliminary experiments, we incubated E/Z-2 with several
OYEs from strains of the genus Saccharomyces, which were able
to reduce 6-membered ring enones. However, those enzymes did
not exhibit the reductase activity toward E/Z-2 (data not shown).
Therefore, enzymes catalyzing reduction of the carbon-carbon
double bond in large monocyclic enones were screened using
E/Z-2 with about 300 yeast and 400 bacterial strains. All bacterial
strains including Pseudomonas putida with morphinone
[
5]
reductase and Bacillus subtilis having OYE family enzyme,
[
9]
YqjM , did not reduce E/Z-2. We found that three yeast strains,
belonging to the genus Sporidiobolus, exhibited high reductase
activity toward E/Z-2. Enzyme from the strain Sporidiobolus
salmonicolor TPU 2001 exhibited the highest reductase activity
(
0.14 mU mg-1 of protein) in the crude enzyme solution. The
reductase, SsERD, was next purified with an overall yield of
2.6% from the strain using four column chromatography
1
Substrate specificity and its kinetic analysis of recombinant
SsERD
experiments (Figure S1, Table 1). The purified enzyme exhibited
a specific activity of 98.2 mU mg-1 of protein with NADPH, but no
activity with NADH (data not shown).
The substrate specificity of SsERD was investigated using E-2, Z-
2, cyclopentadec-2-en-1-one (3), cyclododec-2-en-1-one (4), and
various 5- and 6-membered ring compounds, such as 3-
[
2,
9,
11], cyclohex-2-en-1-one[12],
methylcyclohex-2-en-1-one
Molecular mass and amino acid sequence
ketoisophorone[
3,
12]
,
(R)-(-)-carvone[13], (+)-pulegone[13]
,
2-
Molecular mass of SsERD was estimated to be 80 kDa (data not
shown), which was composed of two identical subunits of 37 kDa
each. The N-terminal amino acid sequence was determined to be
APITNKKTIF, and those of six internal peptides were as follows:
Table 2. Substrate specificity and kinetic parameters of recombinant enone
reductase.
1)
TIFAEVPSGVPEPGK,
2)
TLSLSLDPYLR,
3)
SYVPPFELGQPIANFGTGEVLK, 4) VEVAFNYK, 5) NIFQVVSK,
and 6) GLDNGESFNDLFTGANFGK (Figure S2). The BLAST
search using these amino acid sequences showed that the
identical sequences were present in an uncharacterized protein
from S. salmonicolor CBS 6832[10] (accession number:
CEQ42599).
Substrate
K
µM)
m
V
max
k
cat
-1
k
cat/K
m
-
1
-1
-1
-1
(
(nmol min mg )
(min )
(min µM )
Table 1. Purification of enone reductase from S. salmonicolor TPU 2001.
E-2
Z-2
3
4.9  0.4
100  1.4
3.7  0.1
0.8  0.07
Activity
(mU)
Protein
(mg)
Specific activity
( mU mg )
Yield
(%)
-
1
n.d.[a]
n.d.[a]
n.d.[a]
n.d.[a]
Step
4.3  0.2
1.9  0.3
47  0.6
1.7  0.0
5.9  0.1
0.4  0.02
3.1  0.3
Cell-free extract
Ammonium sulfate
Toyopearl Butyl
Q Sepharose
855
803
340
328
203
108
6104
3210
250
30.2
5.2
0.14
0.25
1.36
10.9
39.0
98.2
100
4
160  3.2
93.9
Each experiment was performed using 3.9 µM–125 µM of substrate under
standard assay condition. Data represent mean values  SD of three
replicates. SsERD did not react with 3-methylcyclohex-2-en-1-one, cyclohex-
-en-1-one,
methylcyclopent-2-en-1-one,
ethylmaleimide, and N-phenylmaleimide. [a] Not detected.
39.8
38.4
23.7
12.6
2
ketoisophorone,
(R)-(-)-carvone,
3-methylcyclopent-2-en-1-one,
(+)-pulegone,
2-
N-
Toyopearl AF-Blue
MonoQ 5/50 GL
1.1
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FULL PAPER
methylcyclopent-2-en-1-one[3, 11], 3-methylcyclopent-2-en-1-one[3,
Effects of pH and temperature on enone reductase activity
and stability
9, 11],
N-ethylmaleimide[12, 14], and N-phenylmaleimide[3, 14] (Table
2
). The enzyme exhibited reductase activity toward E-2, 3, and 4,
When reductase activity of the recombinant SsERD toward E-2
was analyzed in the pH range of 3.0–10.5, the enzyme exhibited
the ERD activity from pH 4.5 to 10.0, and the highest activity was
recorded at pH 8.0 (Figure 2A). The enzyme exhibited optimal
temperature at 45 °C, and the ERD activity drastically decreased
but not toward Z-2 and all small ring compounds. Thus, the
enzyme reacted with 15-membered ring enone with methyl group
and 15- and 12-membered ring enone without methyl group, but
not with 5- and 6-membered ring compounds, which were widely
utilized as the substrates for activity measurements in other ERDs. over 50 °C (Figure 2B). To determine its stability, the enzyme was
The results also indicated that the enzyme was specific to the E-
isomer in 15-membered ring enone bearing a methyl group in the
incubated under varying pH and temperature conditions of 2.5–
13.0 at 30 °C and 10 °C–60 °C at pH 7.0 for 30 min, respectively.
The enzyme was stable in a broad pH range of pH 4.5 to 11.0
(Figure 2C), and up to a temperature of 30 °C, but unstable over
50 °C at pH 7.0 (Figure 2D).

m
-position. The K and Vmax values of SsERD for E-2 were
-1
-1
estimated to be 4.9 ± 0.4 μM and 100  1.4 nmol min mg ,
respectively. The Vmax value for 15-membered ring enone 3 (47 
-1
-1
0
.6 nmol min mg ) was 2 times lower than that of E-2, whereas
the K value (4.3 ± 0.2 μM) was similar to that of E-2. SsERD
exhibited the highest Vmax (160  3.2 nmol min mg ) and affinity
:1.9 ± 0.3 μM) for 12-membered ring enone 4 among the three
m
-1
-1
(K
m
reactive compounds.
Identification of reaction products
To identify the reaction products of three reactive enones E-2, 3,
and 4, each reaction was performed by incubation with the
recombinant SsERD at pH 7.0 at 30 °C. When 0.1 mM of E-2, 3,
or 4 was incubated with 0.2 mM NADPH and 10 mU of SsERD for
60 min in 1 mL of 20 mM potassium phosphate buffer (KPB), they
were converted into S-1, cyclopentadecan-1-one (5), and
cyclododecan-1-one (6) with 98.4%, 97.5%, and 98.8% yields,
respectively, and the ee of S-1 was 100% (Figure 1).
Figure 2. Effects of pH and temperature on enzyme activity and stability. A)
optimal pH, B) optimal temperature, C) pH stability, D) temperature stability.
Error bars indicate the standard deviations of three replicates. ○: KCl-HCl, pH
2
7
.5–3.5, ■: Sodium acetate, pH 4.0–5.5, ●: KPB, pH 6.0–8.0, ▲: Tris-HCl, pH
.5–9.0, ♦: Gly-NaOH, pH 9.0–10.5, □: Na HPO -NaOH, pH 11.0–12.0, ∆: KCl-
2
4
NaOH, pH12.0–13.0
Effects of chemicals and metals on enone reductase activity
One millimolar each of the chemicals and metals was added into
the reaction mixture, and the ERD activity was analyzed under
standard assay conditions using recombinant SsERD. The
enzyme activity was strongly inhibited by mercaptide-forming
reagents such as AgNO
3
,
HgCl
2
,
CuSO
4
,
and p-
chloromercuribenzoic acid, but the activities were recovered by
addition of cysteine into the reaction mixture (Table 3). The ERD
activity was not inhibited by chelating reagents and carbonyl
reagents, and was increased silightly on adding reducing
reagents such as dithiothreitol and cysteine
Effects of site-directed mutagenesis on enzyme activity
A structure model of SsERD was constructed based on the crystal
structure of a double bond reductase from Zingiber officinale
Figure 1. Total ion chromatograms of the reaction products. A) Detection of
muscone. Authentic rac-1 (above), authentic E-2 (middle), and reaction product
(
below). B) Determination of stereochemistry. Authentic R-1 (above), authentic
rac-1 (middle), and reaction product (below). C) Detection of cyclopentadecan-
-one 5. Authentic 5 (above), authentic 3 (middle), and reaction product (below).
(
PDB ID: 4WGG)[15], which showed the highest identity (39.3%)
1
among enzymes with elucidated crystal structure, and four amino
acid residues, Y67, M139, C251, and I281 were selected from its
D) Detection of cyclododecan-1-one 6. Authentic 6 (above), authentic 4 (middle),
and reaction product (below).
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ChemCatChem
10.1002/cctc.201700244
FULL PAPER
putative active site (Figure S3). Then, these residues were
substituted with alanine, and the ERD activity toward E-2 was
analyzed. The Y67A mutant exhibited higher activity than the
recombinant SsERD, whereas M139A and I281A mutants
showed lower activity. C251A mutant did not exhibit the ERD
activity. Then, the kinetic parameters of four mutants for E-2 and
NADPH were analyzed. The Y67A mutant enzyme showed an
morphinone[5] have been widely studied. For example, OYEs from
Saccharomyces carlsbergensis[2] and Candida macedoniensis[12]
catalyze the reduction of 6-membered ring enones, such as
cyclohexen-2-en-1-one,
3-methylcyclohexen-2-en-1-one,
ketoisophorone, and (R)-(-)-carvone. 12-Oxophytodienoate
[
3]
reductases from Lycopersicon esculentum, enone reductases
from Nicotiana tabacum,[11] and YqjM from Bacillus subtilis
catalyze the reduction of 6-membered ring enones and 5-
membered ring enones, such as 2-methylcyclopent-2-en-1-one
and 3-methylcyclopent-2-en-1-one. It was also reported that
some of the above enzymes and N-ethylmaleimide reductase
from Yarrowia lipolytica[ react with N-ethylmaleimide and N-
phenylmaleimide. However, there are no reports of enzymes
catalyzing the reduction of large monocyclic enones, such as 12-
or 15-membered ring enones. Therefore, we screened for
enzymes catalyzing the reduction of large monocyclic enones and
discovered a new enzyme from S. salmonicolor TPU 2001,
SsERD, which catalyzes the reduction of carbon-carbon double
bond of E-2.
[9]
apparent K
nmol min-1 mg-1 for E-2, in which the Vmax value was 4.5 times
higher than that of the recombinant SsERD, while the K value
m
value of 6.6  0.6 µM and Vmax value of 454  0.0
m
was similar to that of the recombinant SsERD (Table4). However,
the apparent Vmax values of M139A and I281A mutants for E-2
14]
were lower than that of the recombinant SsERD, while K
for E-2 were similar to or lower than that of the recombinant
SsERD. Since M139A and I281A mutants showed higher K
m
values
m
values for NADPH (120  7.4 µM and 167  17 µM, respectively),
the low Vmax values of both mutants for E-2 might be affected by
the low affinity to NADPH. In the C251A mutant, ERD activity was
completely lost, indicating that cysteine residue might strongly
affect the SsERD activity.
The SsERD catalyzes the reduction of carbon-carbon double
bond of not only (E)-3-methylcyclopentadec-2-en-1-one (E-2 in
Table 2) but also cyclopentadec-2-en-1-one (3 in Table 2) and
cyclododec-2-en-1-one (4 in Table 2). However, the enzyme did
not catalyze the reduction of (Z)-3-methylcyclopentadec-2-en-1-
one (Z-2 in Table 2), and small ring enones. Thus, SsERD is the
first enzyme catalyzing the reduction of large monocyclic enones.
In addition, the enzyme could recognize the structural difference
between E-2 and Z-2. Since SsERD showed unique substrate
specificity, its other characteristics were further compared in detail
with ERDs catalyzing the reduction of small ring enones.
Discussion
ERDs catalyze reduction of carbon-carbon double bond of α, -
unsaturated ketones,[1] and the enzymes catalyzing the reduction
[
2, 3, 9,
of small ring enones such as 5- or 6-membered ring enones
1
1-14, 16, 17]
or polycyclic enones such as progesterone[4] and
ERDs can be classified into two groups based on their cofactor
specificity: one contains a flavin and NAD(P)H dependent
enzymes such as enzymes of the OYE family,[2, 3, 9, 12, 14] and
Table 3. Effects of chemicals and metals on enone reductase activity.
Compounds
Relative activity (%)
another contains NAD(P)H dependent enzymes such as ERDs
from N. tabacum (NtDBR),[13] guinea-pig leukotriene B4 12-
None
100
97
EDTA
hydroxydehydrogenase/15-oxo-prostaglandin
13-reductase
o-Phenanthroline
101
101
93
(
LTB
4
12-HD/PGR),[16] double bond reductase from Arabidopsis
NH OH
2
thaliana (At5g16970),[17] and double bond reductase from
Zingiber officinale (ZoDBR).[15] Therefore, we searched for
binding site of SsERD using BLAST program, and obtained the
Semicarbazide
Iodoacetic acid
AgNO3
76
0
result that a similar NAD(P) binding site was conserved as in LTB
2-HD/PGR (PDB ID: 1V3V)[16] and human 15-ketoprostaglandin
4
HgCl2
0
1
CuSO4
28
[18]
delta-13-reductase (PDB ID: 2ZB4), which were registered in
PDB as the complexes with NADP. This result indicates that
SsERD is an NADPH dependent enzyme, although the total
amino acid sequence is not so much similar to that of the above
NAD(P)H dependent ERDs (amino acid identity is 35% of NtDBR,
PbCl2
97
N-Ethylmaleimide
97
p-Chloromercuribenzoic acid
42
KCN
100
110
132
90
Cysteine
Dithiothreitol
4
33% of LTB 12-HD/PGR, 38% of At5g16970, and 39% of
ZoDBR).
[
a]
AgNO + Cysteine
The inhibitor specificity and site-directed mutagenesis
suggested that the interaction with C251 residue and NADPH was
important for enzyme activity of SsERD. The enzyme activity of
SsERD was remarkably inhibited by incubation with mercaptide-
forming reagents and recovered on addition of cysteine (Table 3).
The enzyme activity of SsERD was also completely lost with the
mutation of C251 (Table 4). It was reported that ZoDBR (PDB ID:
3
[
a]
HgCl + Cysteine
74
2
[
a]
CuSO + Cysteine
113
117
4
p-Chloromercuribenzoic acid + Cysteine [a]
Each experiment was performed using 1 mM of chemicals or metals
under standard assay condition.
[
a] After addition of 1 mM of chemicals or metals, 2 mM of cysteine was
added to the reaction mixtures, and enzyme activity was assayed under
standard condition.
4
4WGG), LTB 12-HD/PGR (PDB ID: 1V3T), and At5g16970 (PDB
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FULL PAPER
Table 4. Kinetic parameters for E-2 and NADPH of mutant enzymes.
E-2[a]
NADPH[b]
Mutant
K
m
V
max
k
cat
k
cat/K
m
K
m
V
max
k
cat
kcat/K
m
(µM)
(nmol min-1 mg-1)
(min-1)
(min-1 µM-1)
(µM)
(nmol min-1 mg-1)
(min-1)
(min-1 µM-1)
Recombinant
SsERD
4.9  0.4
100  1.4
3.7  0.1
0.8  0.07
8.6  0.7
106  0.0
3.9  0.0
0.5  0.0
Y67A
M139A
C251A
I281A
6.6  0.6
5.3  0.2
n.d.[c]
454  0.0
35  0.2
n.d.[c]
17  0.0
1.3  0.0
n.d.[c]
2.6  0.3
0.2  0.0
n.d.[c]
10  0.6
120  7.4
n.d.[c]
454  0.0
47  0.5
n.d.[c]
17  0.0
1.7  0.0
n.d.[c]
1.6  0.1
0.014  0.001
n.d.[c]
2.7  0.2
5.8  0.2
0.2  0.0
0.08  0.003
167  17
6.8  0.4
0.25  0.01
0.002  0.001
[
[
[
a] Kinetic parameters for E-2 were analyzed using 3.9 µM–125 µM of E-2 and 200 µM NADPH under standard assay condition.
b] Kinetic parameters for NADPH were analyzed using 6.3 µM–200 µM of NADPH and 100 µM E-2 under standard assay condition.
c] Not detected.
Data represent mean values  SD of three replicates.
ID: 2J3I) have cysteine residues in their active sites and interact
with NADPH, although the cysteine residue is not always
conserved in all ERDs.
Experimental Section
The site-directed mutagenesis of Y67, M139, and I281, which
were present in the putative active sites in the homology model,
provided additional information regarding to the substrate
specificity and relationship between NADPH and enzyme activity.
Y67A mutant exhibited 4.5 times higher specific activity toward E-
Chemicals
E/Z-2 was synthesized by Toho Earthtech (Niigata, Japan). E- and Z-
isomers of 2 were separated using the protocol given by Yamamoto et
al.[8c] R-1 was prepared using the protocol of Takabe et al.[20] Compunds 3
and 4 were prepared from 5 and 6, respectively (Figure S4 and S5). All
other chemicals used were of analytical grade and commercially available.
2
, but lower specific activity toward compounds 3 and 4, as
compared with those of the recombinant SsERD (Table S2). The
kinetic analysis of Y67A mutant indicated that the K values for
m
Screening for E/Z-2 reducing enzymes
E-2 and NADPH were similar to those of the recombinant SsERD.
Screening for E/Z-2 reducing enzymes was carried out using yeast strains
of our laboratory at Toyama Prefectural University, Toyama, Japan (TPU).
The strains were cultivated in 10 mL of YPD medium (1% yeast extract,
2% Polypepton, and 2% glucose, pH 7.0) at 30 °C for 24 h, and the cells
were harvested by centrifugation (9,000 × g, 5 min, 4 °C). The cells were
then disrupted using a Multi-beads Shocker instrument (Yasui Kikai,
Osaka, Japan) (2,500 rpm, 60 s on time, 60 s off time, 6 cycles), and cell
debris was discarded after centrifugation (20,000 × g, 20 min, 4 °C). The
supernatant was dialyzed against 20 mM KPB, pH 7.0, and the dialyzed
enzyme solution was used following ERD activity measurement. The
dialyzed enzyme solution containing 1 mg of proteins was incubated with
On the other hand, mutant enzymes of M139A and I281A
exhibited higher K values for NADPH compared with that of the
m
recombinant enzyme. Thus, we revealed that M139 and I281
residues might play an important role in the interaction with
NADPH, and affected to the enzyme activity.
We revealed that SsERD can catalyze the synthesis of large
monocyclic ketones from the corresponding enones. Large
monocyclic ketone derivatives are applicable as synthetic
precursors of bicyclic or polycyclic compounds.[19] Here, we
reported for the first time, an enzyme that catalyzes the reduction
of large monocyclic ketones, and successfully developed new
biochemical method for synthesis of large monocyclic ketones.
1
mM E/Z-2 and 2 mM NADPH in 20 mM KPB (1 mL), pH 7.0, at 30 °C.
After 1 h of incubation, the reaction mixture was vigorously mixed with ethyl
acetate (1 mL), and the products of organic layer were analyzed by GC-
MS to confirm the production of muscone.
Enzyme assay
Conclusions
GC-MS method: The reaction mixture (1 mL) contained 0.1 mM E-2, 0.2
mM NADPH, an appropriate amount of enzyme, and 20 mM KPB, pH 7.0.
The reaction was initiated by adding 2 µL of 50 mM E-2 solution in DMSO,
and incubated at 30 °C for 1 h. The reaction was terminated by adding
ethyl acetate (1 mL), and muscone in organic layer was analyzed by GC-
MS equipped with TC-70 column (φ 0.25 µm, 60 m × 0.25 mm, GL-Science,
Japan). The amount of muscone was quantified using a standard curve of
the authentic muscone. One unit of enzyme activity was defined as the
amount of enzyme catalyzing the formation of 1 µmol of muscone per min.
UV method: The reaction mixture (total volume of 1 mL) containing 0.1
mM E-2, an appropriate amount of enzyme, and 20 mM KPB, pH 7.0, was
incubated at 30 °C for 3 min, and the reaction was initiated by the addition
of 0.2 mM NADPH. The reaction was monitored at 30 °C for 3 min by
measuring absorbance at 340 nm. One unit of enzyme activity was defined
In this study, we found a new enone reductase catalyzing the
reduction of carbon-carbon double bond of large monocyclic
enones. Thus, the enzyme can produce (S)-muscone by
asymmetric reduction of -methyl substituted 15-membered ring
enone. The enzyme also catalyzes the synthesis of
cyclododecan-1-one and cyclopentadecan-1-one from 12- and
15-membered ring enones, which do not possess methyl groups.
Large monocyclic ketone derivatives are also important
intermediates in the synthesis of bicyclic or polycyclic compounds.
The enzyme may be useful for development of new environmental
friendly methods for biochemical synthesis of various large
monocyclic ketones.
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ChemCatChem
10.1002/cctc.201700244
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as the amount of enzyme catalyzing the dehydrogenation of 1 µmol of
NADPH per min.
analysis. The enzyme on SDS-PAGE was destained with 30% acetonitrile
containing 25 mM ammonium hydrogen carbonate, and reductive-
alkylated with iodoacetamide. The trypsin digestion was carried out at
37 °C for 16 h, and the digested peptides were analyzed using a nanoLC-
Analysis of muscone
nanoESI-QTOF (Waters, MA, USA) equipped with a nanoACQUITY
symmetry C18 (φ 5 µm, 180 µm × 20 mm, Waters) as a trap column and
nanoACQUITY CSH 130 C18 (φ 1.7 µm, 75 µm × 200 mm, Waters) as an
analysis column maintained at 35 °C. Elution was carried out with
Muscone was analyzed using the GC-MS system (GC-2010/GCMS-
QP2010 Plus, Shimadzu, Japan) equipped with a TC-70 capillary column.
The column was held at 80 °C for 3 min after injection, and followed by
5.0 °C min-1 increase to 290 °C. The carrier gas used was helium at a
2
-
1
2
H O/acetonitrile containing 0.1% formic acid, 1% (0–1 min), 1–50% (1–50
flow rate of 28 mL min and a column head pressure of 194 kPa was
maintained. The ion source temperature was set at 200 °C in electron
ionization (EI). Muscone and substrate were identified by the m/z value,
and the retention times were compared with authentic standards.
min), 50–95% (50–55 min), 95% (55–75 min), 95–99% (75–78 min)
-
1
acetonitrile, at a flow rate of 0.3 µL min . LC-MS/MS conditions were as
follows: electron ionization, nanoESI; cone voltage, 3 kV; fragmentation,
collision-induced dissociation. The data was acquired by repeating the
collision conditions (10–50 eV per second) and low energy conditions. The
homologous sequences of the internal amino acid sequences obtained
from de novo peptide sequencing were searched by BLASTP.
The stereochemistry of muscone was analyzed using the same
apparatus equipped with cyclodextrin-3P column (φ 0.25 µm, 0.25 mm ×
2
5 m, Chiral-separations com., Germany). The column was held at 80 °C
for 5 min after injection, and followed by 5 °C min-1 increase to 230 °C. The
carrier gas used was helium at a flow rate of 15.2 mL min-1 and a column
head pressure of 150 kPa was maintained. The ion source temperature
was set at 230 °C in EI. Stereochemistry of muscone was identified by
comparison of retention time with authentic standards.
Cloning and expression of the SsERD gene, and purification of the
recombinant enzyme
Total RNA was prepared from the cultivated cells using TRIzol (Thermo
Fisher Scientific, MA, USA) reagent. cDNA was synthesized using
PrimeScriptTM RT-PCR Kit (Takara Bio, Otsu, Japan). The coding region
of the enzyme was amplified by polymerase chain reaction (PCR) using
forward (5’-GGAGATATACCATGGCGCCCATCACCAACAAGAAGACC-
Cultivation of S. salmonicolor TPU 2001
S. salmonicolor TPU 2001 was cultivated in YPD medium (10 mL) at 30 °C
for 24 h. This culture was then inoculated into 500 mL of the YPD medium
and further cultivated at 30 °C for 24 h. The cells were harvested by
centrifugation (8,000 × g, 10 min), washed with 20 mM KPB, pH 7.0, and
stored at -80 °C until use.
3’) and reverse (5’-TGCGGCCGCAAGCTTACTCGAGGGAGATGACGG-
CCTTGCC-3’) primers, which were based on the sequence in accession
number CENE01000030 in the GenBank, with start and stop codons. The
PCR was performed by using 0.23 pmol of forward primer, 0.23 pmol of
reverse primer, 42 ng of gDNA template, and PrimeSTAR MAX premix
Purification of enone reductase from S. salmonicolor TPU 2001
All procedures were performed at 4 °C using KPB, pH 7.0, unless
otherwise stated.
(Takara Bio) in a final volume of 25 µL. PCR conditions were as follows:
35 cycles at 98 °C for 10 s, 55 °C for 15 s, and 72 °C for 8 s. The amplified
PCR products were separated by agarose-gel electrophoresis, purified by
a Wizard SV PCR and Gel Clean-Up System (Promega, WI, USA), and
ligated into pET28a vector using In-Fusion® HD Cloning Kit (Takara Bio).
The DNA sequence was determined using a 3500 Genetic Analyzer
Cells (85 g of wet cell weight from 2 L of culture) were suspended with
320 mL of 20 mM buffer containing 1 mM phenylmethylsulfonyl fluoride
(PMSF), and disrupted using a Multi-beads Shocker (2,500 rpm, 60 s on
time, 60 s off time, 6 cycles). The supernatant (330 mL) was collected after
centrifugation (20,000 × g, 20 min), and 69 g of solid ammonium sulfate
was added into the enzyme solution up to 35% saturation. The resulting
precipitate was removed after centrifugation at 20,000 × g for 20 min, and
(Applied Biosystems, CA, USA).
The plasmid harboring the enone reductase gene was transformed into
E. coli strain BL21 (DE3). The recombinant E. coli was cultivated in 5 mL
of Luria-Bertani (LB) broth containing 20 µg kanamycin/mL for 16 h at
92 g of solid ammonium sulfate was further added to the supernatant up
37 °C. An aliquot (1%) of the grown cells was added into 500 mL of LB
to 75% saturation. The precipitate formed was collected by centrifugation
at 20,000 × g for 20 min and dissolved with 20 mM buffer containing 1 mM
PMSF.
medium containing the same concentration of kanamycin, and incubated
for 4 h at 37 °C. In order to induce the expression, 0.5 mM IPTG was added
into the culture, and the culture was further cultivated for 16 h at 16 °C.
The recombinant enzyme was purified from 30 g of wet cells from 4 L of
culture, using the same procedures as those used for the native enzyme.
Solid ammonium sulfate was added to the enzyme solution to 20%
saturation, and the enzyme solution was applied to a Toyopearl Butyl-650
column (5.5 × 6.0 cm) equilibrated with 20 mM buffer containing 20%
saturated ammonium sulfate. After the column was washed with a buffer
containing 15% saturated ammonium sulfate, the adsorbed enzyme was
eluted with the buffer containing 10% saturated ammonium sulfate. The
active fractions were combined and dialyzed against 20 mM buffer.
The dialyzed enzyme solution was then applied to a Q Sepharose Fast
Flow column (2.5 × 8.0 cm) equilibrated with 20 mM buffer, and
unadsorbed enzymes were collected.
The enzyme solution was applied to a Toyopearl AF-Blue HC-650
column (1.5 × 2.5 cm) equilibrated with 20 mM buffer, and the column was
washed with the same buffer. The adsorbed enzyme was eluted with the
buffer containing 10 mM NADPH, and the active fractions were
concentrated to 3 mL using Amicon Ultra-15 (Merck Millipore, MA, USA).
The concentrated enzyme solution was applied to a MonoQ 5/50 GL
Site-directed mutagenesis of SsERD
Site-directed mutagenesis of the enone reductase gene was performed
using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies,
CA, USA), according to manufacturer’s instruction. Primers were designed
as shown in Table S3, and pET28a vector carrying enone reductase was
used as a template for the mutagenesis reactions. PCR conditions were
as follows: 95 °C for 2 min, 18 cycles at 95 °C for 20 sec, 60 °C for 10 sec,
and 68 °C for 3.5 min, and 68 °C for 5 min.
Effects of pH and temperature on enzyme activity and stability
The optimal pH was assayed by the UV method in the pH range of 4.0–
10.5 using sodium acetate, pH 4.0–5.5, KPB, pH 6.0–8.0, Tris-HCl, pH
7.5–9.0, and glycine-NaOH, pH 9.0–10.5. The optimal temperature was
analyzed at pH 7.0 by varying the temperature between 5 °C and 60 °C.
The pH stability was investigated by incubation at 30 °C for 30 min without
enzyme using KCl-HCl, pH 2.5–3.5, sodium acetate, pH 4.0–5.5, KPB, pH
(GE Healthcare, IL, USA) equilibrated with 20 mM buffer and unadsorbed
active fractions were combined and used for further studies.
Amino acid sequence analysis
6.0–8.0, Tris-HCl, pH 7.5–9.0, glycine-NaOH, pH 9.0–10.5, Na
2 4
HPO -
The N-terminal amino acid sequence was analyzed by Nippi Incorporated
NaOH, pH 11.0–12.0, and KCl-NaOH, pH 12.0–13.0. The temperature
(Tokyo, Japan). The internal sequence was determined by LC-MS/MS
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ChemCatChem
10.1002/cctc.201700244
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stability was analyzed after incubating at 20–65 °C for 30 min in KPB, pH
[6]
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7.0. After the incubation, the remaining activity was analyzed under
standard assay conditions.
[
7]
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a) M. Doya, WO2010109650, 2010; b) Y. Tanabe, N. Matsumoto, T.
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Kanisawa, Tetrahedron 2002, 58, 9209–9212.
The molecular mass of native enzyme was estimated by gel filtration on
a Superdex 200 10/300 GL (GE Healthcare) equilibrated with 20 mM KPB,
pH 7.0, containing 150 mM NaCl. Standard proteins were glutamate
dehydrogenase (290,000 Da), lactate dehydrogenase (142,000 Da),
enolase (67,000 Da), myokinase (32,000 Da), and cytochrome c (12,400
Da).
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Keywords: asymmetric synthesis • enone reductase • large
monocyclic enone • muscone • mutagenesis
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Kazunori Yamamoto, Yuko Oku,
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Purification and characterization of a
novel enone reductase from
Sporidiobolus salmonicolor TPU 2001
reacting with large monocyclic
enones
Substituting the Ru-BINAP catalyst: This is the first report of enone reductase
reducing carbon-carbon double bond of large monocyclic enones. The enone
reductase is available for asymmetric synthesis of (S)-muscone from (E)-3-
methylcyclopentadec-2-en-1-one, which has so far only been reported by chemical
reduction using Ru-BINAP catalyst.
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