Purification and properties of a carbonyl reductase involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate from Cylindrocarpon sclerotigenum IFO 31855

Article abstract of DOI:10.1271/bbb.67.1417

A NADPH-dependent carbonyl reductase (CSCR1) was purified to homogeneity from Cylindrocarpon sclerotigenum IFO 31855. The enzyme catalyzed the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to the corresponding (5)-alcohol with a >99% enantiomer excess. The relative molecular mass of the enzyme was estimated to be 68,000 by gel filtration chromatography and 24,800 on SDS polyacrylamide gel electrophoresis. The enzyme had an extremely narrow substrate specificity and it highly reduced conjugated diketone, 2,3-butanedion, in addition to ethyl 4-chloro-3-oxobutanoate. The enzyme activity was inhibited by HgCl₂ (100%), 5,5′-dithiobis(2-nitrobenzoic acid) (56%), dicoumarol (42%), and CuSO₄ (46%). The N-terminal amino acid sequence of the enzyme (P-Q-G-I-P-T-A-S-R-L) showed no apparent similarity with those of other oxidoreductases.

Full text of DOI:10.1271/bbb.67.1417

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Purification and Properties of a Carbonyl Reductase
Involved in Stereoselective Reduction of Ethyl
4-Chloro-3-oxobutanoate from Cylindrocarpon
sclerotigenum IFO 31855
Yuri SARATANIa, Eiji UHEDAa, Hiroaki YAMAMOTOb, Atsuo NISHIMURAa & Fumiki
YOSHIZAKOa
a Research Institute for Advanced Science and Technology, Osaka Prefecture
University1-2 Gakuen-cho, Sakai, Osaka 599-8570, Japan
b Tsukuba Research Center, Daicel Chemical Industries Ltd.27 Miyukigaoka, Tsukuba-City,
Ibaraki 305-0841, Japan
Published online: 22 May 2014.
To cite this article: Yuri SARATANI, Eiji UHEDA, Hiroaki YAMAMOTO, Atsuo NISHIMURA & Fumiki YOSHIZAKO (2003)
Purification and Properties of a Carbonyl Reductase Involved in Stereoselective Reduction of Ethyl 4-Chloro-3-
oxobutanoate from Cylindrocarpon sclerotigenum IFO 31855, Bioscience, Biotechnology, and Biochemistry, 67:6,
1417-1420, DOI: 10.1271/bbb.67.1417
To link to this article: http://dx.doi.org/10.1271/bbb.67.1417
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Biosci. Biotechnol. Biochem., 67 (6), 1417–1420, 2003
Note
Puriˆcation and Properties of a Carbonyl Reductase Involved in
Stereoselective Reduction of Ethyl 4-Chloro-3-oxobutanoate from
Cylindrocarpon sclerotigenum IFO 31855
1
Yuri SARATANI,1 Eiji UHEDA,1 Hiroaki YAMAMOTO,2 Atsuo NISHIMURA
,
1, ,
†
*
and Fumiki YOSHIZAKO
1Research Institute for Advanced Science and Technology, Osaka Prefecture University, 1-2 Gakuen-cho,
Sakai, Osaka 599-8570, Japan
2Tsukuba Research Center, Daicel Chemical Industries Ltd., 27 Miyukigaoka, Tsukuba-City,
Ibaraki 305-0841, Japan
Received November 29, 2002; Accepted February 24, 2003
A NADPH-dependent carbonyl reductase (CSCR1)
was puriˆed to homogeneity from Cylindrocarpon
sclerotigenum IFO 31855. The enzyme catalyzed the
stereoselective reduction of ethyl 4-chloro-3-oxobutano-
However, very few publications describe the isolation
and characterization of ECAA-reducing enzymes in
other organisms.7,14) In our previous paper,6) we
reported that whole cells of Cylindrocarpon
sclerotigenum IFO 31855 stereoselectively reduced
ate to the corresponding (S)-alcohol with a À99%
enantiomer excess. The relative molecular mass of the
enzyme was estimated to be 68,000 by gel ˆltration
chromatography and 24,800 on SDS polyacrylamide gel
electrophoresis. The enzyme had an extremely narrow
substrate speciˆcity and it highly reduced conjugated
diketone, 2,3-butanedion, in addition to ethyl 4-chloro-
3-oxobutanoate. The enzyme activity was inhibited
ECAA to (
cess (e.e.) and that their cell-free extract also convert-
ed ECAA to ( )-ECHB in the presence of NADPH
e.e. 99 ). For the practical use of fungi for syn-
z
S)-ECHB with a À99 enantiomer ex-
S
(
; À
z
thesis of chiral ECHB, more detailed information on
the properties of the enzymes is needed. In this
paper, we puriˆed the ECAA-reducing enzyme from
C. sclerotigenum IFO 31855 and characterized it
brie‰y.
by HgCl2 (100
%
), 5,5
?
-dithiobis(2-nitrobenzoic acid)
(56 ), dicoumarol (42
%
%
), and CuSO4 (46 ). The N-
%
terminal amino acid sequence of the enzyme (P-Q-G-I-
P-T-A-S-R-L) showed no apparent similarity with those
of other oxidoreductases.
The culture and the preparation of a cell-free
extract of C. sclerotigenum IFO 31855 were as
described previously.6) The enzyme was puriˆed by
four sequential column chromatographies at 0–4
as follows. The standard buŠer, 50 m potassium
phosphate buŠer (KPB, pH 7.0) containing 20
glycerol and 0.5 m DTT, was generally used
9C
Key words: carbonyl
reductase;
Cylindrocarpon
M
sclerotigenum IFO 31855; (S)-ethyl 4-
chloro-3-hydroxybutanoate
z
M
throughout the enzyme puriˆcation. Step 1: The cell-
free extract (250 ml) was put on a DEAE-Toyopearl
Optically active ethyl 4-chloro-3-hydroxybutano-
ate (ECHB) could be used in the synthesis of biologi-
cally and pharmacologically important compounds
650M column (30 mm
×
30 cm, Tosoh, Japan)
equilibrated with the standard buŠer. After washing
of the column with the standard buŠer, the enzyme
was eluted with 700 ml of a linear gradient of NaCl
such as
-carnitine1) and hydroxymethylglutaryl-CoA
L
(HMG-CoA) reductase inhibitor.2) Many studies
have been reported on the asymmetric reduction of
ethyl 4-chloro-3-oxobutanoate (ECAA) to chiral
alcohols using microorganisms.1,3–8) The several kinds
of enzymes capable of reducing ECAA were isolated
from yeasts; baker's yeast,9,10) Sporobolomyces
(0 to 0.6 ) in the standard buŠer. Two peaks show-
M
ing NADPH-dependent ECAA reducing activities
were observed on a DEAE-Toyopearl 650M column
chromatography, one being non-adsorbed (CSCR1)
and the other being eluted at 0.36 to 0.42
(CSCR2). These two peaks gave ( )-ECHB preferen-
tially (e.e. 99 ). On a native polyacrylamaide gel
M
NaCl
11)
12)
salmonicolor
,
Candida
magnoliae
,
and
S
13)
Kluyveromyces lactis
,
and characterized in detail.
;
À
z
†
To whom correspondence should be addressed. E-mail: yoshizako
＠
mua.biglobe.ne.jp
*
Present address: 1-3-305-303 Fujisawadai, Tondabayashi-city, Osaka 584-0071, Japan
Abbreviations: ECAA, ethyl 4-chloro-3-oxobutanoate; ECHB, ethyl 4-chloro-3-hydroxybutanoate; e.e., enantiomer excess; CSCR1,
carbonyl reductase 1 from Cylindrocarpon sclerotigenum

1418
Y. SARATANI et al.
Table 1. Puriˆcation of ECAA Reducing Enzyme (CSCR1)
Total protein
(mg)
Total activity
Speciˆc activity
Overall yield
Yield for CSCR1
Step
*
(U
)
(U mg)
(
z
)
(
z
)
W
Cell-free extract
737
87.4
9.0
3.2
0.91
1,360
903
666
281
114
1.85
10.3
74.0
87.8
125.3
100
—
100
73.8
31.1
12.6
DEAE-Toyopearl
Phenyl Toyopearl
Hydroxyapatite 1st
Hydroxyapatite 2nd
66.4
49.0
20.7
8.4
*
One unit of the enzyme was deˆned as the amount catalyzing the oxidation of 1
m
mol of NADPH per min.
electrophoresis (PAGE), CSCR2 did not enter into
the separation gel but was retained in the stacking
gel. CSCR1 was further puriˆed by sequential
column chromatography as follows; Step 2: The
CSCR1 solution was treated with ammonium sulfate
apparent overall recovery of CSCR1 were 12.6
8.4 respectively. The ECHB formed by CSCR1
was the ( )-enantiomer with 99 e.e. in the
presence of NADPH. The reversibility of the reac-
tion was investigated with (
)-ECHB and NADP＋
using the standard assay. No increase of the absor-
bance at 340 nm was observed. Neither a decrease at
340 nm due to the reduction of ECAA nor ECHB
formation were observed when NADPH was
replaced by an equimolar concentration of NADH.
z
and
z
S
À
z
R,S
at 80
dissolved in 20 ml of the standard buŠer containing
20 (NH4)2SO4 (buŠer A) and dialyzed against the
z
saturation. The resultant precipitate was
z
same buŠer. The enzyme solution was put on a
×
phenyl Toyopearl 650M column (20 mm 25 cm,
Tosoh, Japan) equilibrated with buŠer A. After the
column was washed with buŠer A, the enzyme was
eluted with a linear decrease in the ionic strength
CSCR1 showed the maximum activity at 35
pH 6.5 for the reduction of ECAA in KPB.
9
C and
The N-terminal amino acid sequence of CSCR1
was found to be P-Q-G-I-P-T-A-S-R-L. When this
sequence was compared with those of proteins stored
in NCBI nr, no apparent similarity was found with
other oxidoreductases.
(20 to 0
z
(NH4)2SO4 in standard buŠer, 400 ml).
The fractions showing activities were collected,
concentrated with a Centriprep (YM 10, Millipore),
and dialyzed against 10 m
20 glycerol and 0.5 m
M
KPB (pH 7.0) containing
DTT (buŠer B). Step 3:
z
M
The molecular mass of CSCR1 was estimated to be
The dialyzed solution was put on a Bio Gel HTP
hydroxyapatite column (16 mm 13 cm, Bio-Rad,
68,000 on a Superdex 75 HR 10 30 gel ˆltration
W
×
column (Amersham Pharmacia Biotech.). The
relative molecular mass of the denatured CSCR1
was approximately 24,800.
USA) equilibrated with buŠer B. The enzyme was
eluted with 200 ml of linear gradient of KPB (10 to
400 m
M
, pH 7.0). The fractions showing activities
The substrate speciˆcity of CSCR1 is shown in
Table 2. CSCR1 showed restricted substrate speciˆci-
ty. The enzyme had high activities for ECAA but no
were concentrated with a Centriprep and dialyzed
against buŠer B. Step 4: The dialyzed enzyme
solution was put on a 2nd hydroxyapatite column as
described above. The enzyme solution was concen-
trated with a Centriprep and then used for characteri-
zation of the enzyme. The enzyme activity (standard
activity for other
b-keto esters or cyclohexanone.
Low activities for ethyl pyruvate and hydroxyacetone
were also observed. Interestingly, CSCR1 eŠectively
reduced conjugated diketone, 2,3-butanedione and
2,3-pentanedione but 2,4-pentanedione was not
reduced. The enzyme did not catalyze the reduction
of typical substrates for aldo-keto reductase family
assay) was measured at 359C by measuring the rate
of decrease in absorbance at 340 nm as described
previously.6) One unit of the enzyme was deˆned as
the amount catalyzing the oxidation of 1
m
mol
enzyme, such as
p-nitrobenzaldehyde, pyridine-3-
NADPH per minute. Protein was measured by the
protein-dye binding method15) using bovine serum
albumin as a standard. The amino acid sequence of
the enzyme was analyzed by the Edman method with
a Perkin Elmer Model 491-1 protein sequencer. Poly-
aldehyde, or DL-glyceraldehyde. Aldoses, such as
D
(
＋
)-glucose,
D
(
＋
)-galactose, or
D
( )-xylose which
＋
are good substrates for aldehyde- and aldose-reduc-
tases, were not also reduced by the enzyme.
The eŠects of various chemicals on the activity of
CSCR1 are shown in Table 3. Among the sulfhydoryl
acrylamide (5 to 20
z
) gel electrophoresis (PAGE)
and sodium dodecyl sulfate (SDS)-PAGE were done
by the methods of Davis16) and Laemmli,17) respec-
tively.
inhibitors tested, neither
um -chloromercuribenzoate inhibited the enzyme
activity, but 5-5 -dithiobis(nitrobenzoate) inhibited
the activity around 56 . HgCl2 inhibited it complete-
ly but CuSO4 and ZnSO4 inhibited the activity
around 46 and 0 respectively. These results
indicate that CSCR1 resists SH-blocking reagents.18)
N-ethylmaleimide nor sodi-
p
?
The puriˆcation of CSCR1 is summarized in
Table 1. The puriˆed CSCR1 gave a single band on
both SDS-PAGE and native PAGE. The recovery of
CSCR1 after the DEAE Toyopearl step and the
z
z
z

Carbonyl Reductase from Cylindrocarpon sclerotigenum
1419
Table 2. Substrate Speciˆcity of CSCR1
chain alcohol dehydrogenase reductases (S1 and S4)
W
from Candida magnoliae have been character-
ized.12,23) The molecular masses of S1 and S4 are
77,000 and 86,000 as the native form, and 32,000 and
29,000 as the subunit form, respectively. These
enzymes also have narrow substrate speciˆcity, and
do not reduce typical substrates for aldo-keto reduc-
tases, i.e. p-nitrobenzaldehyde, pyridine-3-aldehyde,
or aldoses. The molecular mass of CSCR1 from C.
sclerotigenum are 68,000 as the native form and
24,800 as the subunit form. CSCR1 seems to be a
dimer or trimer. The properties of CSCR1, the
molecular mass and narrow substrate speciˆcity,
resembled those of S1 and S4. CSCR1 may be a
member of the short-chain alcohol dehydrogenase
Relative
activiaty
Substrate
m
M
(
z)
Ethyl 4-chloro-3-oxobutanoate
Ethyl 3-oxobutanoate
Methyl 3-oxopentanoate
Ethyl 2-methyl-3-oxobutanoate
Cyclohexanone
Ethylpyruvate
Hydroxyacetone
2,3-Butanedione
2,3-Pentanedione
2,4-Pentanedione
33.3
33.3
33.3
33.3
33.3
33.3
33.3
33.3
33.3
33.3
1
100
5
0
0
0
14.6
12.5
106
22.7
0
0
0
0
0
0
0
0
0
3.2
0
o
m
-Nitrobenzaldehyde
-Nitrobenzaldehyde
1
1
1
1
p
o
m
-Nitrobenzaldehyde
-Chlorobenzaldehyde
-Chlorobenzaldehyde
W
reductase group. However, the N-terminal amino
acid sequence of CSCR1 shows no similarity with
those of S1, S4, and other oxidoreductases. Thus
CSCR1 seems to be a novel NADPH-dependent
carbonyl reductase. This work is thus the ˆrst to
demonstrate the puriˆcation and properties of a car-
p
-Chlorobenzaldehyde
1
Pyridine-3-aldehyde
DL-Glyceraldehyde
33.3
33.3
33.3
33.3
33.3
D
D
D
-Xylose
-Galactose
-Glucose
0
bonyl reductase from the genus of Cylindrocarpon
.
However, to ˆnd whether this enzyme can be used
practically, further biochemical studies are needed.
a
To calculate the relative activity, the activity with 33.3 m
taken as 100
M
ECAA was
z.
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C before the
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