Purification and characterization of two alpha-keto ester reductases from Streptomyces thermocyaneoviolaceus IFO 14271.

Article abstract of DOI:10.1271/bbb.66.588

Two NADPH-dependent alpha-keto ester reductases (Streptomyces thermocyaneoviolaceus keto ester reductase, STKER-II and -III) were purified from S. thermocyaneoviolaceus IFO 14271, one of thermophilic actinomycetes. The molecular masses of native STKER-II

Full text of DOI:10.1271/bbb.66.588

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Purification and Characterization of Two α-
Keto Ester Reductases from Streptomyces
thermocyaneoviolaceus IFO 14271
Hitomi YAMAGUCHI, Nobuyoshi NAKAJIMA & Kohji ISHIHARA
To cite this article: Hitomi YAMAGUCHI, Nobuyoshi NAKAJIMA & Kohji ISHIHARA (2002)
Purification and Characterization of Two α-Keto Ester Reductases from Streptomyces
thermocyaneoviolaceus IFO 14271, Bioscience, Biotechnology, and Biochemistry, 66:3, 588-597,
DOI: 10.1271/bbb.66.588
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Date: 07 November 2015, At: 23:55

Biosci. Biotechnol. Biochem., 66 (3), 588–597, 2002
Puriˆcation and Characterization of Two a-Keto Ester Reductases
*
from Streptomyces thermocyaneoviolaceus IFO 14271
3,
†
Hitomi YAMAGUCHI,1 Nobuyoshi NAKAJIMA,2 and Kohji ISHIHARA
1Institute for Chemical Research, Kyoto University, Uji 611-0011, Japan
2Department of Nutritional Science, Graduate School of Health and Welfare,
Okayama Prefectural University, Soja, Okayama 719-1197, Japan
3Department of Chemistry, Kyoto University of Education, Fushimi-ku, Kyoto 612-8522, Japan
Received October 1, 2001; Accepted November 9, 2001
15)
16,17)
Two NADPH-dependent
Streptomyces thermocyaneoviolaceus keto ester reduc-
a-keto ester reductases
ii
,
Geotrichum candidum
,
and Klebsiella pneu-
(
moniae18) that can also catalyze the asymmetric
reduction of keto esters are also used for the prepara-
tion of chiral hydroxy esters. However, little infor-
mation is known from mechanistic studies of en-
zymatic reduction by their microorganisms.13,14,19,20)
To date, we have studied the microbial reduction
tase, STKER-II and -III) were puriˆed from S. thermoc-
yaneoviolaceus IFO 14271, one of thermophilic ac-
tinomycetes. The molecular masses of native STKER-II
and -III were estimated to be 60 kDa and 70 kDa by gel
ˆltration chromatography, respectively. These enzymes
were both homodimers, with 29-kDa and 30-kDa subu-
nit molecular masses based on SDS polyacrylamide gel
electrophoresis. STKER-II and -III were stable from pH
7.0 to 10.0 and pH 5.5 to 9.0, respectively. Ethyl 3-
methyl-2-oxobutanoate was reduced by both enzymes
of some a- and b-keto esters with thermophilic Bacil-
lus strains, actinomycetes, and micro algae and have
reported that some of their strains could reduce keto
esters with high conversion and excellent enantiomer-
ic excess (e.e.).21–25) In particular, we found that the
isolated to the corresponding (R)-hydroxy ester with ex-
stereoselectivity of
a-keto ester reduction using a
cellent enantiomeric excess. STKER-III showed high
stereoselectivity for the reduction of bulky substrates,
while the selectivity of the STKER-II-catalyzed reduc-
tion was low except for ethyl 3-methyl-2-hydrox-
ybutanoate. Both enzymes had small Km values toward
aliphatic keto esters having a long alkyl chain.
thermophilic actinomycete, S. thermocyaneov-
iolaceus IFO 14271, changed with alterations in the
reaction conditions such as reaction temperature,
substrate concentration, and introduction of addi-
tives.23) For example, ethyl 3-methyl-2-oxobutanoate
was reduced by S. thermocyaneoviolaceus to the cor-
responding (
while the reduction at 55
responding ( )-hydroxy ester. In order to elucidate
the reaction mechanism, we tried to isolate -keto
R
)-hydroxy ester with high e.e. at 37
9
C,
Key words: thermophilic actinomycete; reduction;
9
C produced the cor-
reductase; enzyme puriˆcation;
ester producing enzyme
a
-hydroxy
S
a
ester-reducing enzymes from a cell-free extract of S.
thermocyaneoviolaceus IFO 14271; consequently,
one enzyme (STKER-I) was puriˆed and character-
Biotransformations of exogenous substrates have
been widely studied to synthesize chiral com-
pounds.1–6) The microbial reduction enables prepara-
tion of optically pure alcohols and helps to lessen the
environmental impact of organic syntheses. For ex-
ample, the asymmetric reduction of keto esters by
bakers' yeast has been widely used to obtain chiral
compounds, because the produced hydroxy esters are
useful chiral building blocks for the synthesis of
natural and bioactive compounds.7–9) Furthermore,
several yeast keto ester reductases (YKERs) were
isolated from bakers' yeast and their enzymatic
properties studied including the speciˆc activity,
stereoselectivity, and kinetic parameters.10–14) Other
microorganisms such as Thermoanaerobactor brock-
ized. The enzyme reduced various
corresponding (
)-hydroxy ester with excellent e.e.26)
However, ( )-hydroxy ester-producing enzyme(s)
a-keto esters to the
S
R
were still not isolated from the cells.
We would like to report here the puriˆcation and
characterization of new two a-keto ester reductases
from S. thermocyaneoviolaceus IFO 14271. We will
also discuss the reaction mechanism in the actinomy-
cete cells on the basis of enzyme properties.
Materials and Methods
Chemicals. p-ABSF (4-(2-aminoethyl)benzenesul-
*
Stereoselective reduction of
To whom correspondence should be addressed. FAX: +81-75-645-1734; E-mail: kishi
a
-keto esters with thermophilic actinomycete, Part 4.
†
＠
kyokyo-u.ac.jp

a
-Keto Ester Reductases from Thermophilic Actinomycete
589
fonate ‰uoride) and ethyl pyruvate were purchased
from Wako Pure Chemicals, Japan. Ethyl 3-methyl-
2-oxobutanoate was obtained from Aldrich Chemi-
cals, USA. Methyl benzoylformate and ethyl ben-
zoylformate were purchased from Tokyo Kasei
sion was cooled at 09C, then sonicated with 15 pulses
of 120 sec each with 300-sec cooling intervals in a
Sonicator (Ohtake Works, Japan), ˆtted with a
microtip at a power setting of 70 W. At the ˆrst inter-
val,
ˆnal concentration was 0.5 m
homogenate was removed by centrifugation at 10,000
p
-ABSF was added as a protease inhibitor (the
Kogyo, Japan. Other
a
-keto esters were synthesized
M
). Cell debris in the
according to methods from the literature.27) DTT
(dithiothreitol) and Bis-tris propane (1,3-bis[tris
(hydroxymethyl)methylamino]propane) were ob-
tained from Nacalai Tesque, Japan. NADPH and
NADH were obtained from Kohjin, Japan. EDTA
×
g
9
for 30 min at 4 C, and the supernatant served as
the crude cell-free extract.
Puriˆcation of STKER-II.
Step 1. DEAE-Toyopearl chromatography. The
cell-free extract (176 ml) was applied to a DEAE-
(ethylenediaminetetraacetic acid) and MES (2-(
N
-
morpholino)ethanesulfonic acid) were purchased
from Dojindo Laboratories, Japan. Extrelut was
purchased from Merck, Germany. All other chemi-
cals used in this study were of analytical grade and
commercially available.
Toyopearl 650
M
×
(TOSOH, Japan) column (q 7.0
8.5 cm) previously equilibrated with BuŠer-A. After
the column was washed with BuŠer-A (600 ml), the
protein was eluted with a 0 to 0.3
M
increasing linear
gradient of KCl in BuŠer-A (800 ml). The fractions
Microorganism and cultivation. S. thermoc-
yaneoviolaceus IFO 14271 was purchased from the
IFO (Institute for Fermentation, Osaka) culture
collection, Japan. The actinomycete was cultivated
containing the enzyme activity (eluted at about 0.2
KCl) were pooled.
M
Step 2. AF-Red-Toyopearl column chromato-
graphy. The above enzyme solution (236 ml) without
concentration or a desalting procedure was applied to
9
aerobically at 45 C for 15 h. The cells were harvested
by ˆltration in vacuo and washed with saline as
described previously.26)
a
AF-Red-Toyopearl 650 ML (TOSOH, Japan)
×
q 3.2 3.0 cm) equilibrated with BuŠer-A.
column (
Enzyme assay. The reducing activity of each reduc-
tase (STKER-II and -III) was measured spec-
trophotometrically using ethyl 2-oxoheptanoate as
After washing with BuŠer-A, the protein was eluted
with a 0 to 3.0
BuŠer-A (200 ml). All fractions containing activity
(eluted about 1.5 ) were collected (90 ml), concen-
M
increasing linear gradient of KCl in
the substrate (ˆnal concentration was 4.0 m
2.0 m , respectively). The standard assay mixture
(1.0 ml) comprised 0.1 potassium phosphate buŠer
(KPB) (pH 6.5), 0.15 m NADPH, substrate
M
and
M
M
trated to 21 ml using a Stirred Cells Model 8400
(Amicon Grace, Japan) with an ultraˆltration mem-
brane YM 10 (Dia‰o, cutoŠ MW 10,000), and dia-
lyzed overnight against BuŠer-A.
M
M
solution, and enzyme solution. The consumption of
reduced coenzyme was followed with a Beckman
DU-640 spectrophotometer at 340 nm at 37
unit of the enzyme activity was deˆned as the amount
of enzyme catalyzing the oxidation of 1 mol
NADPH min under the conditions speciˆed.
9
C. One
Step 3. POROS HS column chromatography. The
dialyzate solution was applied to a POROS HS
m
(Applied Biosystems, USA) column (q 2.0 1.6 cm)
×
equilibrated with BuŠer-A. After the column was
washed with BuŠer-A, the protein was eluted with a
W
Protein assay. The protein content was measured
by the method of Bradford,28) calibrated with
-lac-
0.1 to 0.3
BuŠer-A (150 ml). All fractions containing activity
(eluted between 0.2 and 0.25 ) were collected
M
increasing linear gradient of KCl in
g
toglobulin as a standard (Bio-Rad Protein Assay
Kit).
M
(16 ml), concentrated to 9.8 ml by a Stirred Cells
Model 8050 (Amicon Grace, Japan) equipped with
an ultraˆltration membrane YM 10, and dialyzed
overnight against BuŠer-A.
Enzyme puriˆcation. All puriˆcation procedures
were done below 4
BuŠer-A: 20 m
taining 1 m DTT, 1 m
(pH 7.2). BuŠer-B: BuŠer-A containing 1.3
nium sulfate (pH 7.2). BuŠer-C: BuŠer-A containing
0.2 KCl (pH 7.2).
9C unless otherwise speciˆed.
M
potassium phosphate buŠer con-
EDTA, and 10 glycerol
ammo-
M
M
z
Step 4. Mono Q HR10 10 column chromato-
W
M
graphy. The enzyme solution was applied to a Mono
Q
HR10 10 (Amersham Pharmacia Biotech,
W
M
Sweden) column equilibrated with BuŠer-A (‰ow
rate was 1.5 ml min). The protein was eluted with a
W
Preparation of the cell-free extract. The cells were
ground under cooled acetone (below „30 C) and
0.2 to 0.4
M
increasing linear gradient of KCl in
9
BuŠer-A. The fractions containing the reducing
activity (3.0 ml) were collected and concentrated to
0.5 ml by a Centricon-30 (cutoŠ MW 30,000, Amicon
Grace, Japan).
ˆltered in vacuo (for preparing acetone-powder of
the cells). Fifty-eight grams of the powdered cells
were suspended in 550 ml of BuŠer-A. This suspen-

590
H. YAMAGUCHI et al.
Step 5. Asahipack GS 520 column chromato-
0.7 ml min). The active fraction was pooled and
W
graphy. The concentrated enzyme solution was ap-
used as the puriˆed enzyme for characterization.
plied to an Asahipack GS 520 (Showa Denko, Japan)
×
gel ˆltration column (
q
7.6 300 mm) equilibrated
Determination of molecular mass of enzyme. The
molecular masses of the native STKER-II and -III
were estimated by high performance liquid column
chromatography (HPLC) analysis with Asahipack
with BuŠer-C (‰ow rate was 0.7 ml min). The active
fraction was pooled and used as the puriˆed enzyme
for characterization.
W
×
GS 520 (q 7.6 300 mm) and Superdex 200 HR 10
W
Puriˆcation of STKER-III.
Step 1. DEAE-Toyopearl chromatography. The
cell-free extract (252 ml) was applied to a DEAE-
30 columns, respectively, with a standard molecular
marker (Oriental Yeast, Japan) with BuŠer-C. The
molecular mass of the subunit was estimated by SDS-
M
column (
q
7.0 6.0 cm) previously
×
poly acrylamide gel erectrophoresis (12.5z) with
Toyopearl 650
equilibrated with BuŠer-A. After the column was
washed with BuŠer-A (600 ml), the protein was elut-
SDS-PAGE marker (Bio-Rad, USA) as the stan-
dard.29)
ed with a 0 to 0.2
in BuŠer-A (600 ml). All fractions containing the
reducing activity (eluted at about 0.1 KCl) were
M
increasing linear gradient of KCl
Measurement of kinetic parameters. The Km and
Vmax of the puriˆed enzymes toward various a-keto
M
collected (168 ml) and concentrated to 53 ml by a
Stirred Cells Model 8400 equipped with an ultraˆltra-
tion membrane YM 10.
esters and NADPH were calculated from the initial
rates of the reaction in an appropriate range of sub-
strate concentrations using at least ˆve points by
Lineweaver-Burk plots.30)
Step 2. AF-Red-Toyopearl column chromato-
graphy. The concentrated solution was applied to a
Measurement of the eŠects of pH. The optimum
pH of the enzyme (relative activity) was measured at
various pHs in the following buŠers: pH 5.5 to 7.5,
×
q 3.2 3.0 cm)
AF-Red-Toyopearl 650 ML column (
equilibrated with BuŠer-A, and another portion of
BuŠer-A was then allowed to ‰ow into the column.
The void fraction (not adsorbed) was collected and
pooled (68 ml).
0.1
5.5 to 7.0, 0.1
buŠer; pH 6.0 to 7.5, 0.1
M
KPB; pH 7.5 to 9.0, 0.1
Citrate-K2HPO4 and 0.1
Bis-tris propane buŠer.
M
Tris-HCl buŠer; pH
M
M
MES
M
The relative activity was calculated by arbitrarily set-
ting the activity at pH 6.0 to be 100. The pH stability
of the enzyme (residual activity) was measured at
Step 3. Butyl-Toyopearl column chromatography.
To the above enzyme solution was added 11.9 g of
ammonium sulfate powder (adjusted pH 7.2 with
NH3 aq.), and then the mixture was stirred for 2 h at
37
10 min in the following buŠers: pH 5.0 to 7.5, 0.1
KPB; pH 8.0 to 8.5, 0.1 Tris-HCl buŠer; pH 9.0 to
11.0, 0.1 glycine-KOH buŠer. The residual activity
9C in 0.1
M
KPB (pH 6.5) after incubation for
M
0
9
C. The mixture was put on a Butyl-Toyopearl
650 4.0 6.0 cm) column equilibrated with
BuŠer-B. The column was washed with BuŠer-B, and
the protein was eluted with a 1.3 to 0 (240 ml)
decreasing linear gradient of (NH4)2SO4. The active
fractions (eluted at about 0.3 ) were collected
M
M
(q
×
M
was calculated by setting the activity without incuba-
tion to be 100.
M
M
EŠects of additives. The enzyme (residual) activity
(43 ml), concentrated to 10 ml by a Stirred Cells
Model 8050 equipped with an ultraˆltration mem-
brane YM 10, and dialyzed overnight against BuŠer-
A.
was measured in 0.1
M
9
KPB (pH 6.5) at 37 C after a
5-min incubation in the buŠer in the presence of each
additive (metal ion, chelator, sulfhydryl reagent, or
arginine-speciˆc reagent). The residual activity was
calculated by arbitrarily setting the activity in the ab-
sence of any additives to be 100.
Step 4. Mono Q HR10 10 column chromato-
W
graphy. The dialyzate solution was applied to a
Mono Q HR10 10 column equilibrated with BuŠer-
Enzymatic reduction of
propylene tube were placed the puriˆed enzyme solu-
tion (1.0 unit), NADPH (10 mol), the substrate
(10 mol), and 0.1 KPB (pH 7.0, 0.5 ml). The mix-
ture was shaken gently at 37 C. After 2 h, the mix-
a-keto esters. In a poly-
W
A (‰ow rate was 2.0 ml min). The protein was eluted
W
with a 0 to 0.2
M
increasing linear gradient of KCl in
m
BuŠer-A. The active fraction (eluted at about 0.13
M
)
m
M
was collected (5.2 ml) and concentrated to 0.6 ml by
a Centricon-30.
9
ture was ˆltered by an Extrelut short column, extract-
ed with ether, and then concentrated under reduced
pressure.
Step 5. Superdex 200 HR10 30 column chromato-
W
graphy. The enzyme solution was applied to a
Superdex 200 HR 10 30 (Amersham Pharmacia
Stereochemistry of the products. The enantiomeric
excesses of ethyl lactate, ethyl 2-hydroxy butanoate,
ethyl 2-hydroxy pentanoate, ethyl 2-hydroxy hexano-
W
×
q 1.0 30
Biotech, Sweden) gel ˆltration column (
cm) equilibrated with BuŠer-C (‰ow rate was

a
-Keto Ester Reductases from Thermophilic Actinomycete
591
Table 1. Puriˆcation of STKER-II from S. thermocyaneoviolaceus IFO 14271
Total protein
(mg)
Total activity
(units)
Speciˆc activity
Puriˆcation
Step
Yield (
100
35.6
30.1
27.7
17.0
z)
(units mg)
fold (
z)
W
Cell-free extract
DEAE-Toyopearl
AF-Red-Toyopearl
POROS HS
Mono Q
Asahipack GS520
9970
1520
69.4
10.3
0.465
0.137
1920
682
577
531
326
158
0.192
0.448
8.31
51.6
701
1
2.33
43.2
268
3640
5980
1150
8.24
Table 2. Puriˆcation of STKER-III form S. thermocyaneoviolaceus IFO 14271
Total protein
(mg)
Total activity
(units)
Speciˆc activity
Puriˆcation
Step
Yield (
100
35.1
7.62
z)
(units mg)
fold (
z
)
W
Cell-free extract
DEAE-Toyopearl
AF-Red-Toyopearl
Butyl-Toyopearl
Mono Q
14500
897
480
124
10.7
0.178
2660
933
203
141
64.6
37.0
0.184
1.04
0.422
1.13
6.04
208
1
5.66
2.30
6.15
5.29
2.43
1.39
32.9
1130
Superdex 200
ate, ethyl 2-hydroxy heptanoate, ethyl mandelate,
and methyl mandelate were determined by GLC
(GL Science GC-353 gas chromatograph) analyses
(Chirasil-DEX CB, Chrompack, Netherlands,
×
0.25 mm 25 m, 85 to 1309C), and that of ethyl
2-hydroxy-3-methylbutanoate was also measured
by GLC analysis (Chiraldex G-TA, Astec, USA,
0.25 mm
×
25 m, 95 C). The absolute conˆguration
9
of the isomer was identiˆed by comparing its reten-
tion time with those of authentic samples prepared
according to the method in the literature.27)
Fig. 1. SDS-PAGE of the Puriˆed Enzymes.
SDS-gel electrophoresis using 12.5 polyacrylamide was
done in the presence of 0.1 SDS. (A, C, E) Standards (from
top): phosphorylase b (Mr 97,400), bovine serum albumin
(66,200), hen egg white ovalbumin (45,000), carbonic anhydo-
lase (31,000), soybean trypsin inhibitor (21,500), and hen egg
white lysozyme (14,400). (B) STKER-II. (D) STKER-III. Pro-
teins were stained with Coomassie Brilliant Blue R-250 and
N-Terminal amino acid sequence analysis. The N-
terminal amino acid sequence was analyzed on a
model 476A pulsed liquid protein sequencer (Applied
Biosystems, USA) with an on-line phenylthiohydan-
toin amino acid analyzer.31) The N-terminal se-
quences obtained were compared with those of pro-
teins stored in protein sequence databases (GenBank,
EMBL, PIR, and SWISS-PROT) using the sequence
similarity search programs, BLAST and FASTA.32,33)
z
z
＝
destained in 30
(v v) water.
z (v v) methanol 10z (v v) acetic acid 60z
W W W W
W
Molecular mass and subunit structure
Results and Discussion
The relative molecular masses of the native
STKER-II and -III were estimated to be 60 kDa and
70 kDa, respectively, by gel ˆltration column chro-
matography. The molecular masses of the subunit
were also estimated to be 29 kDa and 30 kDa, respec-
Puriˆcation of STKER-II and STKER-III
Two a-keto ester reductases, STKER-II and -III,
were puriˆed via ˆve chromatographic steps from a
cell-free extract of S. thermocyaneoviolaceus IFO
14271 to homogeneity with a 8.2
z
and 1.4
z
overall
z
tively, by SDS-PAGE (12.5 ). Two a-keto ester
recovery, respectively. The puriˆcation of STKER-II
and -III is summarized in Tables 1 and 2. Each of the
puriˆed enzymes gave a single band on SDS-PAGE
(Fig. 1). The speciˆc activity of STKER-II was
reductases puriˆed in this study had a similar size
(molecular mass of the native and the subunit); fur-
thermore, the size of the two enzymes was similar to
that of STKER-I from the same actinomycete.26)
1150 units mg with 4.0 m
M
ethyl 2-oxoheptanoate,
W
and that of STKER-III was 208 units mg with
Substrate speciˆcity
The relative activities of the puriˆed enzymes
W
2.0 m
M
ethyl 2-oxoheptanoate.

592
H. YAMAGUCHI et al.
Table 3. Substrate Speciˆcity of STKER-II and STKER-III form S. thermocyaneoviolaceus IFO 14271a
b
b
b
b
Rel. rate (
STKER-II
z
)
Rel. rate (
STKER-II
z
)
Rel. rate (
STKER-II
z
)
Rel. rate (
STKER-II
z
)
Substrate
Substrate
Substrate
Substrate
-III
36
-III
39
-III
3
-III
0
5
20
42
81
100
53
59
7
4
25
7
0
0
0
0
60
100
70
71
73
0
c
—
19
7
0
0
0
17
0
0
c
7
—
29
0
69
0
0
2
0
0
0
59
0
3
c
8
—
0
34
0
4
43
0
0
0
0
0
a
b
c
Concentration was 5 m
Rel. rate Relative rates were calculated by setting the activity to be 100 (STKER-II: ethyl 2-oxoheptanoate, STKER-III: ehyl 2-oxopentanoate).
Not measured.
M for a-keto esters and 20 mM for other substrates.
＝
toward various keto esters and other carbonyl
compounds were examined spectrophotometrically.
These results are summarized in Table 3. STKER-II
Stereospeciˆcity
The reduction of
a
-keto esters by the puriˆed en-
zymes was carried out to clarify the stereochemistry
of the products, as shown in Table 4. The enan-
tioselectivity of the reduction by STKER-II was not
had greater activity toward aliphatic
a-keto esters
having a long alkyl chain such as 2-oxohexanoate and
2-oxoheptanoate, while the activity for smaller sub-
strates such as ethyl pyruvate and methyl pyruvate
was low. STKER-III showed high activity not only
for aliphatic substrates but also for aromatic sub-
strates. STKER-II had the reducing acitivity for ethyl
as precise (from 46
for ethyl 3-methyl-2-oxobutanoate (
and ethyl 2-oxobutanoate (86 e.e. (
reduced -keto esters having a long alkyl chain, an
isopropyl or a phenyl group to the corresponding
)-hydroxy esters with excellent e.e. (such as ethyl
z
e.e. (
S
) to 40
z
À
e.e. (
99
R)). STKER-III
R
)) except
z
e.e. ( ))
R
z
a
4-chloroacetoacetate in
b
-keto esters we tested.
-keto
(R
STKER-III also had activities toward some
b
2-oxoheptanoate, ethyl 3-methyl-2-oxobutanoate,
ethyl benzoylformate, and methyl benzoylformate),
while the selectivity for ethyl pyruvate and 2-
esters (ethyl 2-chloroacetoacetate, ethyl 2-allylaceto-
acetate, ethyl 4-chloroacetoacetate, methyl acetoa-
cetate, ethyl acetoacetate, ethyl 2-methyl acetoa-
cetate, and ethyl benzoylacetate) slightly. The
reducing activities by STKER-II and -III for other
substrates, such as benzyl acetoacetate, 1-acetoxy-2-
propanone, 1-methoxy-2-propanone, acetophenone,
oxobutanoate was low (14
tively) and gave the (
z
and 38
z
e.e., respec-
S)-hydroxy esters.
Kinetic parameters
The kinetic constants (Km and
V
max) for aliphatic
propiophenone, 2
none, and (-)-menthone were not observed. Both
enzymes showed reducing activity toward neither
keto acids such as pyruvic acid, 3-methyl-2-
oxobutanoic acid, and mandelic acid nor -nitroben-
?
,3
?
,4
?
,5
?
,6
?
-penta‰uoroacetophe-
and aromatic a-keto esters were calculated by
Lineweaver-Burk plots as shown in Table 5. The
Km of STKER-II toward ethyl pyruvate, ethyl 2-
oxobutanoate, ethyl 2-oxopentanoate, ethyl 2-
oxohexanoate, and ethyl 2-oxoheptanoate were 32.1,
a
-
p
zaldehyde, a typical substrate for aldo-keto reduc-
tases.
21.6, 4.48, 2.45, and 0.831 m , respectively. As the
M
alkyl chain in the substrate molecule became longer,
the corresponding Km of STKER-II was decreased.
On the contrary, the Vmax tended to become larger
with extension of the substituent. Thus, STKER-II
prefers substrates having a long alkyl chain.
These enzymes were highly speciˆc for NADPH as
a sole coenzyme. The oxidation activity for ethyl lac-
tate and ethyl 2-hydroxy-3-methyl butanoate was not
found for either enzyme.

a
-Keto Ester Reductases from Thermophilic Actinomycete
593
STKER-III showed strong reducing ability for
aromatic -keto esters. The Vmax Km for ethyl
benzoylformate was larger than those of STKER-I
(1.41 mol min mg m ) and -II in the actinomycete
a
W
W W M
m
W
cells. These results suggest that STKER-III contrib-
utes mainly to reduction of ethyl benzoylformate to
give the corresponding (
tinomycete cells.
R)-a-hydroxy ester in ac-
Optimum pH and pH stability
The eŠects of pH on the enzyme activity were exa-
mined as shown in Fig. 2. Both of the two puriˆed
enzymes showed maximum activity about pH 6.0–6.5
Table 4. Stereoselectivity of the Enzymatic Reductiona,b
STKER-II
STKER-III
Substrate
e.e. (
z
)
(
R S
)
e.e. (
z
)
(
R S
)
W
W
40
R
R
R
S
14
S
S
86
37
34
46
99
31
15
38
81
71
99
99
99
99
S
R
R
R
R
R
Fig. 2. The Optimum pH and pH Stability of the Enzyme.
The pH dependence (relative activity) was measured in 0.1
buŠer (pH 5.5 to 11.0) at 37 C (line graph). ( ) Potassium
) Tris-HCl buŠer, ( ) Citrate-K2HPO4
) MES buŠer were
S
À
À
À
À
M
9

À
R
R
S
$
phosphate buŠer, (

#
buŠer, ( ) Bis-tris propane buŠer, and (

used. The relative activity was calculated by arbitrarily setting
the activity at pH 6.5 to be 100. The pH stability (residual activi-
ty) was measured in 0.1
cubation for 10 min in 0.1
graph). The residual activity was calculated by setting the activi-
M
potassium phosphate buŠer after in-
buŠer (pH 5.5 to 11.0) at 37 C (bar
M
9
a
The puriˆed enzyme solution (1.0 unit), NADPH (10
(10 mol), and 0.1 potassium phosphate buŠer (pH 7.0, 0.5 ml) were
incubated for 2 h at 37 C.
Enanitiomeric excesses and conˆguration were measured by GLC analy-
sis with optically active capillary columns.
mmol), substrate
m
M
ty without incubation to be 100. (A) STKER-II. (B) STKER-III.
9
b
Table 5. Kinetic Parameters of STKER-II and STKER-III from S. thermocyaneoviolaceus IFO 14271
STKER-II
STKER-III
mol min mg)
Substrate
Km (m
32.1
M
)
Vmax
(
m
mol min mg)
Vmax Km
Km (m
4.05
M
)
Vmax
(
m
Vmax Km
W
W
W
W
W
W
11.2
0.350
55.8
228
156
38.5
156
751
272
622
38.4
341
697
21.6
1210
1.47
230
218
258
250
312
212
227
4.48
2.45
0.831
2.99
4.95
1020
1550
1720
1170
415
0.290
0.948
0.402
8.12
635
2070
383
83.8
0.621
0.326
a
a
a
—
—
—
a
Not measured.

594
H. YAMAGUCHI et al.
Table 6. EŠects of Additives on STKER-II and STKER-III from S. thermocyaneoviolaceus IFO 14271a
Residual activity (
z
)
z
Residual activity ( )
Additive
None
Metal ions
NaCl
(Conc.)
Additive
(Conc.)
STKER-II
100b
STKER-III
100b
STKER-II
STKER-III
Chelators
-Phenanthroline
2,2 -Bipyridyl
o
(1 m
(1 m
(1 m
M
M
M
)
)
)
95
96
100
101
100
103
(5 m
(5 m
(5 m
(5 m
(5 m
(5 m
(5 m
(5 m
(5 m
M
M
M
M
M
M
M
M
M
)
)
)
)
)
)
)
)
)
96
88
99
94
103
89
82
55
8
110
107
98
104
0
24
16
0
?
MgCl2
KCl
CaCl2
MnCl2
CoCl2
NiCl2
EDTA
Sulfhydryl reagents
p
-CMBc
(1 m
(1 m
M
M
)
)
71
75
62
73
Iodoacetic acid
Arginine-speciˆc reagentsd
Phenylglyoxal
(1 m
M
)
75
78
40
45
ZnCl2
HgCl2
1,2-CHDOe
(15 mM)
0
a
b
c
The enzyme was incubated with additive for 5 min at 37
The activity of each enzyme without inhibitor was calculated by arbitrarily setting to be 100.
9
C before the reaction was started.
＝
p-Chloromercurybenzoate
p
-CMB
Measured after incubation for 15 min at 37
1,2-CHDO 1,2-Cyclohexanedione.
d
e
9
C.
＝
in potassium phosphate buŠer. STKER-II and -III
were stable in the pH 7.0 to 10.0 region and pH 5.5 to
9.0 region, respectively. While STKER-II was unsta-
ble below pH 6.0 and stable under alkaline condi-
tions, STKER-III was stable in acidic regions and un-
stable above pH 9.5.
Thermostability
The thermostability of STKER-II and STKER-III
was investigated by measuring the residual activity
after incubation at 37
Fig. 3. Both enzymes retained 50–60
ty after 4 days of incubation at 37 C. The stability of
STKER-II, STKER-III, and STKER-I were similar
under incubation at 37 C. After incubation at high
temperature (STKER-II and -III: 45 C, STKER-I:
50 C), STKER-II and STKER-III were deactivated
9
C and 45
9
z
C as shown in
of their activi-
9
Fig. 3. Thermostability of the Puriˆed Enzymes.
The stability of the enzyme activity (residual activity) was
measured in 0.1 potassium phosphate buŠer (pH 6.5) at 37
after incubation at 37 C or 45 C in BuŠer-A. After the incuba-
tion, the enzyme activity was immediately assayed. The residual
activities were the values expressed as percent relative to the ac-
9
M
9C
9
9
9
9
for several hours. However, STKER-I did not lose
activity at all for 4 hours at 509
C.26)

tivity without incubation. ( ) STKER-II incubated at 37
9
C,
C, ( ) STKER-II incubated at
C.
#

(
) STKER-III incubated at 37
C, ( ) STKER-III incubated at 45
9
$
45
9
9
Inhibition study
The eŠects of a broad range of metal ions, chela-
tors, sulfhydryl-protecting and -inhibiting reagents,
and arginine-speciˆc reagents on the activity of the
two puriˆed enzymes were investigated (see Table 6).
CHDO) inhibited the activities of both enzymes. This
result indicates an essential arginine residue for the
catalytic activity.
Among the sulfhydryl inhibitors tested, p-chloromer-
curybenzoate and iodoacetic acid inhibited the reduc-
ing activity of both enzymes. STKER-II was inhibit-
ed by mercury and zinc ion. STKER-III was also
inhibited by heavy metal ions, such as manganese,
cobalt, nickel, zinc, and mercury. These inhibition
patterns indicate an essential sulfhydryl group. Metal
N-Terminal amino acid sequences
The N-terminal amino acid sequences of three a-
keto ester reductases, STKER-I (was puriˆed again as
described previously26)), -II, and -III from S. ther-
mocyaneoviolaceus were analyzed by an automated
Edman degradation using a pulsed liquid phase pro-
tein sequencer. The sequences of the N-terminal ami-
no acid of STKER-I, -II, and -III were 1Ala-Thr-His-
Val-Ile-Thr-Gly-Ala-Gly-10Ser-Gly-Ile-Gly-Ala-
Ala-, 1Thr-Ser-Val-Glu-Leu-Pro-Glu-Leu-Ser-
10Gly-Lys-Val-Ala-Leu-Val-, and 1Met-Lys-Arg-
ion chelating reagents such as
o-phenanthroline,
2,2 -bipyridyl, and EDTA showed no inhibitory
?
eŠects on the acitivities. This suggests that both
enzymes do not contain an essential metal ion.
Furthermore, arginine-residue-speciˆc reagents such
as phenylglyoxal and 1,2-cyclohexanedione (1,2-

a
-Keto Ester Reductases from Thermophilic Actinomycete
595
Leu-Val-Thr-Val-Val-Thr-10Gly-Gly-Ser-Arg-Gly-
Ile-, respectively as shown in Fig. 4 (the N-terminals
of the three enzymes were not blocked). All the en-
of S. thermocyaneoviolaceus IFO 14271. The reduc-
tion of ethyl 3-methyl-2-oxobutanoate by the whole
9
cells at 37 C gave the corresponding (R)-hydroxy es-
zymes we puriˆed had
a
consensus sequence
ters.23) This result suggests that the substrate was
(-Gly-X-X-X-Gly-X-Gly-), the putative coenzyme
(NAD(P)H) binding site, in the N-terminal regions
(underlined). The similarity of each sequence of the
three enzymes with those of other proteins was exa-
mined by a computer search of the protein sequence
databases. Among STKER-I and ProSc, STKER-II
and PscoSc, STKER-III and PuoSc, a high homolo-
reduced mainly by STKER-II (the lowest Km and the
highest
Vmax of the three reductases) in the actinomy-
cete cells. However, the stereoselectivity of the ac-
tinomycete reduction was low at high temperature.23)
It is presumed that this stereoselectivity change may
be attributed to diŠerences in the thermostability of
the reductases which contribute to the reduction in
the cells, that is, because the thermostability of
STKER-I is higher than that of STKER-II and -III,
STKER-I contributes to the reduction at high tem-
perature; consequently, the stereoselectivity sifted
gy (92, 92, and 73
z, respectively) was observed.
These homologous proteins were putative or proba-
ble oxidoreductases in the cells of Streptomyces
coelicolor A3(2). These results suggest that STKER-
I, -II, and -III belong to an oxidoreductase family.
9
toward (S)-hydroxy ester production at 55 C. The
stereoselectivity change in the reduction of ethyl
pyruvate, ethyl benzoylformate, and methyl benzoyl-
formate would be explicable in the same manner.
Comparison with other microbial reductases
Several
a-keto ester-reducing enzymes have
been isolated from microorganisms such as Sac-
The isolation of a-keto ester reductase from S. coe-
charomyces cerevisiae (bakers' yeast) and Candida
19)
magnoliae
.
The enzymatic properties of these en-
zymes are compared with those of three enzymes in
the cells of S. thermocyaneoviolaceus IFO 14271 as
shown in Table 7. Although other enzymes including
STKER-II and -III used NADPH as a sole coenzyme,
STKER-I utilized both NADPH and NADH as the
coenzyme. STKER-I had high a‹nity for ethyl pyru-
vate among the listed reductases. Furthermore,
STKER-I reduced ethyl 3-methyl-2-oxobutanoate to
the corresponding (
S)-hydroxy ester in excellent e.e.
(
À
99 e.e.). Therefore, STKER-I would be very
z
useful for catalysis of the preparation of ethyl (
methyl-2-hydroxybutanoate.
S)-3-
Fig. 4. Sequence Comparison of N-Terminal Amino Acid Se-
quences of -Keto Ester Reductases from S. thermocyaneov-
iolaceus and Other Oxidoreductases.
ProSc: Probable oxidoreductase from S. coelicolor A3(2),
accession number: T35808. PscoSc: Putative short chain ox-
idoreductase from S. coelicolor A3(2), accession number:
AL359949, gene: 2SC2G61.27c. PuoSc: Putative oxidoreduc-
tase from S. coelicolor A3(2), accession number: AL359949,
gene: 2SC2G61.17c. The underlined sequences are putative
coenzyme biding sites.
a
STKER-II and -III reduced ethyl 3-methyl-2-
oxobutanoate to the corresponding ( )-hydroxy es-
ter with excellent e.e. as did YKER-IV from bakers'
yeast.
Finally, we examined the mechanistic interpreta-
tion about the stereoselectivity change in the reduc-
tion of ethyl 3-methyl-2-oxobutanoate by whole cells
R
Table 7. Comparison of the Characteristics of the Reductases from Various Microorganisms
Molecular mass
Enzyme (Source)
Coenzyme
CFCa
SDS-PAGE
(kDa)
Km (m
M
)
e.e. (R S
)
Km (m
M
)
e.e. (R S)
W
W
(kDa)
STKER-I26)
STKER-IIb
STKER-IIIb
64
60
70
58
31
83
76
30 (dimer)
29 (dimer)
30 (dimer)
29 (dimer)
39 (monomer)
41 (dimer)
32 (dimer)
0.079
32.1
4.05
135
0.434
5.06
À
99 (
S
R
S
S
R
S
)
)
)
)
)
)
9.01
2.99
À
99 (
S
R
R
)
)
)
NADH NADPH
W
40 (
14 (
98 (
99 (
94 (
À
À
99 (
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
8.12
99 (
YKER-II (Bakers' yeast)14)
N.R.c
N.R.c
YKER-IV (Bakers' yeast)14)
À
0.265
79.4
240
À
99 (
77 (
R
S
)
)
YKER-V (Bakers' yeast)14)
19)
d
d
Carbonyl reductase (Candida magnoliae
)
21
—
—
a
＝
GFC Gel ˆltration chromatography.
b
c
Puriˆed in this study.
N.R. indicates no reaction.
Not determined.
d

596
H. YAMAGUCHI et al.
A., Stereochemical control of microbial reduction.
17. Mechanism to control the enantioselectivity in the
reduction with bakers' yeast. J. Org. Chem., 56,
4778–4783 (1991).
licolor A3(2) and the comparison of enzymatic
properties including the N-terminal amino acid se-
quence of the reductase with those of STKER are
now in progress in our laboratory.
13) Nakamura, K., Kawai, Y., Miyai, T., Honda, S.,
Nakajima, N., and Ohno, A., Stereochemical control
in microbial reduction. 18. Mechanism of
stereochemical control in diastereoselective reduction
with bakers' yeast. Bull. Chem. Soc. Jpn., 64,
1467–1470 (1991).
Acknowledgments
The work was partially supported by the Ministry
of Education, Science, Sport and Culture of Japan,
through a Grant-in-aid for Scientiˆc Research,
Encouragement of Young Scientists, No. 13780452
(2001–2002). We wish to express our appreciation to
Prof. N. Tokitoh, Institute for Chemical Research,
Kyoto University, for his useful advice during the
course of the work.
14) Nakamura, K., Kondo, S., Kawai, Y., Nakajima, N.,
and Ohno, A., Puriˆcation and characterization of a-
keto ester reductases from bakers' yeast. Biosci.
Biotechnol. Biochem., 58, 2236–2240 (1994).
15) Sonnleitner, B. and Fiechter, A., Application of
immobilized cells of Thermoanaerobium brockii for
stereoselective reductions of oxo-acid esters. Appl.
Microbiol. Biotechnol., 23, 424–429 (1986).
16) Biosson, D., Azerad, R., Sanner, C., and Larche-
veque, M., A study of the stereocontrolled reduction
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