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Previous Article | Next Article 
Applied and Environmental Microbiology, October 1999, p. 4399-4403, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Purification and Characterization of Monovalent Cation-Activated
Levodione Reductase from Corynebacterium aquaticum
M-13
Masaru
Wada,1,*
Ayumi
Yoshizumi,1
Shigeru
Nakamori,1 and
Sakayu
Shimizu2
Department of Bioscience, Fukui Prefectural
University, 4-1-1 Kenjyojima, Fukui 910-1195,1
and Division of Applied Life Sciences, Graduate School of
Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto
606-8502,2 Japan
Received 24 May 1999/Accepted 20 July 1999
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ABSTRACT |
(6R)-2,2,6-Trimethyl-1,4-cyclohexanedione (levodione)
reductase was isolated from a cell extract of the soil isolate
Corynebacterium aquaticum M-13. This enzyme catalyzed
regio- and stereoselective reduction of levodione to
(4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone (actinol). The relative molecular mass of the enzyme was estimated to
be 142,000 Da by high-performance gel permeation chromatography and
36,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme required NAD+ or NADH as a cofactor, and it
catalyzed reversible oxidoreduction between actinol and levodione. The
enzyme was highly activated by monovalent cations, such as
K+, Na+, and NH4+. The
NH2-terminal and partial amino acid sequences of the enzyme showed that it belongs to the short-chain alcohol
dehydrogenase/reductase family. This is the first report of levodione reductase.
| |
INTRODUCTION |
Optically active hydroxy
cyclohexanone derivatives, such as
(4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone
(actinol), are useful chiral building blocks of naturally occurring
optically active compounds, such as xanthoxin (3) and
zeaxanthin (11).
Microbial production of actinol from
2,2,6-trimethylcyclohexanedione has been demonstrated previously
by Nishii et al. (15, 16), because actinol is the key
intermediate in zeaxanthin formation. However, in this case, a
racemic mixture of 4-hydroxy-2,2,6-trimethylcyclohexanones was
obtained as the reduction product; this mixture contained (4R,6S)-, (4S,6R)-,
(4R,6R)-, and
(4S,6S)-4-hydroxy-2,2,6-trimethylcyclohexanones at a quantitative ratio of 68:25:5:2, and actinol accounted
for only 5% of the total isomer content. Moreover, the enzyme involved in reduction of 2,2,6-trimethylcyclohexanedione has not been purified yet.
We screened the microorganisms that can catalyze stereo- and
regioselective reduction of the carbonyl group at the C-4 position of
levodione and found that the soil isolate Corynebacterium
aquaticum M-13 was the best producer of the enzyme under the
conditions tested. The enzyme produced actinol with a 95% enantiomeric
excess as the reduction product (Fig. 1).
We describe here purification and characterization of this levodione
reductase from C. aquaticum M-13, which we found to be a
novel, monovalent cation-activated enzyme.
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FIG. 1.
Conversion of
(6R)-2,2,6-trimethyl-1,4-cyclohexanedione to
(4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone by
C. aquaticum M-13.
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MATERIALS AND METHODS |
Microorganisms and cultivation.
Microorganisms preserved in
the AKU Culture Collection (Faculty of Agriculture, Kyoto University)
and soil isolates which can grow on a medium containing
1,4-cyclohexanedione as the sole carbon source were subjected to
screening. The strains were cultivated aerobically at 30°C for
20 h in a medium (pH 7.0) containing 1% glucose, 1.5% peptone,
0.3% K2HPO4, 0.1% yeast extract, 0.2% NaCl, and 0.02% MgSO4 · 7H2O.
Chemicals.
Levodione and actinol were prepared as described
previously (11, 15). 3,5,5-Trimethyl-2-cyclohexene-1,4-dione
(ketoisophrone) and dihydro-3-oxo-4,4-dimethyl-2-furanone (ketopantoyl
lactone) were synthesized as described previously (11, 20).
2,3-Pentanedione and 1-phenyl-1,2-propanedione were purchased from Wako
Pure Chemicals (Osaka, Japan) and Tokyo Kasei Industries (Tokyo,
Japan), respectively. All other chemicals used in this study were of
analytical grade and were commercially available.
Screening of levodione-reducing strains.
Washed cells of
each microorganism obtained from 5 ml of culture broth were incubated
in 1 ml of reaction mixture containing 100 µmol of potassium
phosphate buffer (pH 7.0), 904 nmol of NAD+, 784 nmol of
NADP+, 14.3 U of glucose dehydrogenase (Amano
Pharmaceutical, Nagoya, Japan), 278 mmol of glucose, and 32.5 nmol of
levodione. The reaction mixture was incubated at 30°C for 18 h
with shaking, and then the mixture was vigorously shaken with 1 ml of
ethyl acetate. The ethyl acetate layer was analyzed to determine its
levodione and actinol contents by gas chromatography (GC) as described below.
Analysis of levodione and actinol.
Quantitative analysis of
the levodione and actinol contents was performed with a Shimadzu model
GC-14B GC equipped with a flame ionization detector by using a type
HR-20M capillary column (0.25 mm by 30 m; Shinwa Chemical
Industries, Kyoto, Japan) at 160°C (isothermal) and He as the carrier
gas at a flow rate of 1 ml/min. Under these conditions, levodione,
actinol, and
(4S,6R)-hydroxy-2,2,6-trimethylcyclohexanone (a
diastercomer of actinol) eluted at 6.8, 15.6, and 15.9 min, respectively.
Enzymatic preparation of actinol.
Enzymatic reduction of
levodione was carried out as follows. A 3.3-ml reaction mixture
containing 830 µmol of potassium phosphate buffer (pH 7.0), 1.5 U of
the enzyme, and 130 µmol of NADH was incubated at 30°C. At 5-min
intervals for 30 min, 5 µmol of levodione was added. After 30 min of
incubation, the reaction mixture was extracted with 2 ml of ethyl
acetate, and the ethyl acetate layer was concentrated. The concentrated
ethyl acetate layer was analyzed by GC under the conditions described above.
Enzyme assay.
Enzyme activity was determined by
spectrophotometrically measuring the levodione-dependent decrease in
the NADH content. The standard 2.5-ml assay mixture contained 5 µmol
of levodione (final concentration, 2.0 mM), 0.80 µmol of NADH, 500 µmol of potassium phosphate buffer (pH 7.0), and the enzyme. One unit
of enzyme activity was defined as the amount of enzyme that catalyzed
oxidation of 1 µmol of NADH per min.
When the effect of monovalent cations was measured, the potassium
phosphate buffer was replaced by an equimolar concentration of Tris-HCl
buffer (pH 7.4).
Kinetic studies.
Steady-state kinetic studies were performed
in 100 mM Tris-HCl buffer (pH 7.4). When the apparent
Km value for levodione was determined, the
levodione concentration was varied from 2.0 to 20 mM in the presence of
a fixed concentration of NADH (320 µM). When the apparent
Km value for NADH was determined, the NADH
concentration was varied from 40 to 320 µM in the presence of a fixed
concentration of levodione (10 mM).
The kinetic parameters of the reverse reaction were also determined
with same substrate concentrations.
Purification of the enzyme.
All purification procedures were
performed at 0 to 4°C in 10 mM potassium phosphate buffer (pH 7.0)
containing 0.1 mM dithiothreitol, unless otherwise specified.
The washed cells (wet weight, 60 g) isolated from 8 liters of
culture broth were suspended in 180 ml of the buffer and then disrupted
with an ultrasonic oscillator for 60 min. After centrifugation, the
resulting supernatant was fractionated with solid ammonium sulfate. The
precipitate obtained with 40 to 60% saturation was collected, dialyzed
against 10 liters of the buffer for 18 h, and applied to a
DEAE-Sepharose FF (Pharmacia Biotech, Uppsala, Sweden) column (3.0 by
20 cm) equilibrated with the buffer. The enzyme was eluted with a
linear 0 to 1.0 M NaCl gradient in 660 ml of the buffer at a flow rate
of 2.0 ml/min. The enzyme eluted at approximately 0.28 M NaCl in the buffer.
The concentration of (NH4)2SO4 was
adjusted to 2 M by adding solid
(NH4)2SO4 to the enzyme solution
before the enzyme solution was loaded onto an Alkyl Superose HR10/10
(Pharmacia Biotech) column (1.0 by 10 cm) that was connected to a fast
protein liquid chromatography system (Pharmacia Biotech) and previously
had been equilibrated with the buffer containing 2 M
(NH4)2SO4. The enzyme was eluted
with a linear 2 to 0 M (NH4)2SO4
gradient in 230 ml of the buffer at a flow rate of 0.8 ml/min. The
activity-containing fractions, which eluted at approximately 1.5 M
(NH4)2SO4 in the buffer, were
pooled and dialyzed against 3 liters of the buffer for 8 h.
The enzyme solution was applied to a MonoQ HR5/5 column (0.5 by 5 cm)
that was connected to a fast protein liquid chromatography system and
previously had been equilibrated with the buffer. The enzyme was eluted
with a linear 0 to 0.8 M NaCl gradient in 35 ml of the buffer at a flow
rate of 1 ml/min. The activity-containing fractions, which eluted at
approximately 0.4 M NaCl in the buffer, were collected and used as the
purified enzyme for characterization.
Lysyl endopeptidase digestion and isolation of the peptides.
The purified enzyme was digested with lysyl endopeptidase (Wako Pure
Chemicals, Osaka, Japan) under the conditions described previously
(10). The peptides were separated by reverse-phase high-performance liquid chromatography on a µRPC
C2/C18 column (Pharmacia Biotech) connected to
a Smart system (microscale protein purification system; Pharmacia
Biotech). The peptides were eluted with a linear 0 to 80% acetonitrile
gradient containing 0.1% trifluoroacetic acid.
Amino acid sequence analysis.
A partial amino acid sequence
was determined with a model 476A pulsed liquid protein sequencer
(Applied Biosystems, Foster City, Calif.) as described previously
(22). The partial amino acid sequence obtained was compared
with the sequences of proteins stored in the SWISS-PROT (release
37.0+/06-14, June 99), PIR (release 60.0, March 99), and PRF (release
99-05, May 99) protein databases. Sequence alignment was performed by
using the Blast (1) and Fasta (18) programs.
Other methods.
The molecular mass of the enzyme and protein
concentrations were determined as described previously (22).
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RESULTS |
Screening of levodione-reducing strains.
Levodione-reducing
activity was widely distributed in various microorganisms. As shown in
Table 1, bacteria belonging to the genera
Corynebacterium, Cellulomonas, and
Arthrobacter catalyzed stereoselective reduction of
levodione and gave actinol as a reduction product. One strain, C. aquaticum M-13, was selected as the best producer of the enzyme
that catalyzes the stereoselective reduction of levodione to actinol
under the conditions tested.
Purification of the enzyme.
The enzyme activity of the cell
extract was too low to determine the specific activity; however, after
ammonium sulfate precipitation, the enzyme activity seemed to increase,
which allowed quantitative measurement. The method used for
purification of levodione reductase is summarized in Table
2. The enzyme was purified to
homogeneity, and the overall level of recovery was 10.7%. The purified
enzyme produced a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Fig.
2).
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FIG. 2.
SDS-PAGE of the levodione reductase from C. aquaticum M-13. Lane a, Mr standards,
including (from top to bottom) phosphorylase b
(Mr, 94,000), bovine serum albumin (67,000),
ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor
(20,100), and  -lactalbumin (14,400); lane b, purified enzyme (4 µg). The gel was stained for protein with Coomassie brilliant blue
R-250 and was destained in methanol-acetic acid-water (7:6:47).
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Relative molecular mass and subunit structure.
Using a
calibrated column of Superdex-200 PC3.2/30 (Pharmacia Biotech), we
estimated that the relative molecular mass of the enzyme was 155,000 Da, while the molecular mass determined by high-performance gel
permeation chromatography performed with a Cosmosil 5Diol-300 column
(Nacalai Tesque, Kyoto, Japan) was about 142,000 Da. The relative
molecular mass of each subunit was estimated to be approximately 36,000 Da by SDS-PAGE (Fig. 2). These results suggest that the enzyme is
a tetramer.
Stereoselectivity for levodione reduction.
Using levodione as
the substrate, we analyzed the optical purity of the reduction product
by GC. The actinol formed by the enzyme was the
(4R,6R) enantiomer with 95.0% enantiomeric excess.
Effect of monovalent cations on enzyme activity.
The purified
enzyme eluted from the MonoQ HR5/5 column, which contained
approximately 400 mM NaCl, had a specific activity of 20.7 U/mg.
However, the specific activity of the enzyme decreased to 0.69 U/mg
after overnight dialysis against 10 mM Tris-HCl buffer (pH 7.4)
supplemented with 0.1 mM dithiothreitol.
The activity of the dialyzed enzyme was restored when a monovalent
cation, such as NH4+, Na+,
Cs+, K+, and Rb+, was added to the
reaction mixture. Activation by the monovalent cations K+,
Na+, and NH4+ is shown in Fig.
3.
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FIG. 3.
Effects of K+ ( ), Na+ ( ),
and NH4+ ( ) on enzyme activity. Tris-HCl (pH
7.4) was used as the buffer, and the enzyme activity with 2 M
NH4+ was defined as 100%. Chloride was used as
the anion in all mixtures.
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Substrate specificity and catalytic properties.
The substrate
specificity of the levodione reductase of C. aquaticum M-13
is shown in Table 3. Some cyclic ketones,
including ketoisophorone and ketopantoyl lactone, and some vicinal
diketones, including 2,3-pentanedione and 1-phenyl-1,2-propanedione,
were reduced by this enzyme in addition to levodione. However, other cyclic ketones, including isophrone, cyclohexanone, cyclopentanone, and
1,4-cyclohexanedione, and some typical substrates for aldo-keto reductase, such as p-nitrobenzaldehyde and
pyridine-3-aldehyde, were not reduced. Normal hyperbolic kinetics were
observed with levodione. The apparent Km and
Vmax values under the conditions described
above, both in the presence and in the absence of KCl, as calculated
from Lineweaver-Burk plots, are shown in Table
4.
The enzyme was very specific for NADH as a coenzyme; when the levodione
concentration was 2 mM, the apparent Km value
for NADH was 154 µM, and no decrease was observed at 340 nm due to reduction of levodione when NADH was replaced by an equimolar concentration of NADPH.
The reversibility of the reaction was investigated by using actinol and
NAD+ at concentrations of 2.0 and 0.32 mM, respectively. We
observed an increase in the absorbance at 340 nm due to oxidation of
actinol. When the ethyl acetate extract of the reaction mixture
obtained after oxidation was examined by GC, a peak corresponding to
the levodione peak was observed. Thus, the enzyme catalyzed a
reversible reaction between levodione and actinol. The apparent
Km and Vmax values with
320 µM NAD+ in the presence of 1 M KCl for actinol
oxidation were 1.36 ± 0.25 mM and 15.9 ± 1.2 µmol/min/mg
of protein, respectively.
Partial amino acid sequence analysis.
The
NH2-terminal amino acid sequence of the enzyme was
determined by automated Edman degradation by using a pulsed liquid phase sequencer (Fig. 4). This sequence
was found to be similar to the NH2-terminal amino acid
sequences of the short-chain dehydrogenase/reductase (SDR) family
(4, 19) proteins, such as biphenyl-2,3-dihydro-2,3-diol dehydrogenase of Pseudomonas sp. strain KKS102
(7), cis-1,2-dihydrobenzene-1,2-diol dehydrogenase of Pseudomonas putida (9),
cis-toluene dihydrodiol dehydrogenase of P. putida F1 (24), and
2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase of
Sphingomonas paucimobilis (14),
which belong to the SDR family (4, 19). Moreover, this
NH2-terminal amino acid sequence contains G-X-X-X-G-X-G,
which is highly conserved in the NH2-terminal regions of
SDR family proteins (19). These results strongly suggest
that the enzyme purified in this study belongs to the SDR family.
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FIG. 4.
Comparison of the NH2-terminal and internal
(K-1 and K-2) amino acid sequences of the levodione reductase of
C. aquaticum M-13 (LERE) with the amino acid sequences of
2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase of S. paucimobilis (13) (LinC).
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The enzyme protein was digested with lysyl endopeptidase, and the
resulting digest was separated by the Smart system. Three peptides
(K-1, K-2, and K-3) were isolated, and the amino acid sequences of
these peptides were analyzed with a protein sequencer. The K-1 and K-2
sequences are shown in Fig. 4, and the K-3 sequence was
A-A-V-L-E-T-A-P-D-A-E-V-L-T-T. When these sequences were compared with
the sequences in three protein sequence databases (PIR, PRF, and
SWISS-PROT) by using the sequence similarity search programs Blast
(1) and Fasta (18), the K-1 and K-2 sequences
were found to be significantly similar to partial amino acid sequences of 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase of
S. paucimobilis (14) and 3-oxoacyl-[acyl-carrier
protein] reductase of Haemophilus influenzae
(6), respectively, both of which belong to the SDR family.
However, K-3 did not exhibit significant similarity to any other SDR
family protein. Overall, the partial amino acid sequence of this enzyme
exhibited the greatest similarity to the sequence of
2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase of S. paucimobilis (14). A sequence alignment is shown in
Fig. 4.
Spectral properties.
The absorption spectrum of the enzyme had
a maximum at 278 nm. No absorbance was detected at wavelengths greater
than 320 nm. Thus, the enzyme does not contain flavin or
pyrroloquinoline quinone, which are the coenzymes present in most
quinone reductases (5) and in quinoprotein dehydrogenase
(2).
Effects of temperature on enzyme activity and stability.
As
shown in Fig. 5A, the optimum temperature
for levodione reduction was determined to be 20°C at pH 7.0. Although
the dialyzed enzyme was stable at temperatures below 15°C for 10 min
at pH 7.0, in the presence of 1 M KCl the enzyme was stable at
temperatures below 40°C for 10 min at pH 7.0 (Fig. 5B). At 55°C,
all of the initial enzyme activity was lost under both conditions.
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FIG. 5.
Effects of temperature on enzyme activity (A) and
stability (B). (A) Activity of the enzyme was measured at various
temperatures from 20 to 60°C at pH 7.0. Relative activity is
expressed as percentages of the maximum activity under the experimental
conditions. (B) Enzyme (30 µg), in a total volume of 1.0 ml, was
incubated in 10 mM Tris-HCl buffer (pH 7.4) containing 0.1 mM
dithiothreitol at various temperatures for 10 min in the presence ()
or absence ( ) of KCl. The residual activity was measured under the
standard assay conditions.
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DISCUSSION |
Levodione-reducing microorganisms have been described in previous
papers (15, 16); however, levodione reductase was not purified previously. To our knowledge, this is the first report concerning levodione reductase.
Judging from the substrate specificity (Table 3), a diketone structure
seems to be necessary in order for a compound to be transformed by
levodione reductase. However, the physiological substrate of this
enzyme is still unknown.
This enzyme was activated by monovalent cations, such as
K+, Na+, and NH4+, as
are the glycerol dehydrogenases of Escherichia coli
(21), Aerobacter aerogenes (12), and
Cellulomonas sp. (17, 23). These glycerol
dehydrogenases exhibit maximum activity at monovalent cation
concentrations of 0.7 to 40 mM, although the enzyme purified in our
experiment exhibited maximum activity in the presence of 1.5 to 2.0 M
NH4Cl and 1.8 to 2.0 M NaCl. Oxidoreductases, which exhibit
maximum activity at extremely high salt concentrations, have been found
in some halophilic bacteria, such as Halobacterium salinarium and Halobacterium cutirubrum (8).
However, C. aquaticum M-13 is not a halophilic bacterium and
scarcely grows in the presence of 1 M KCl.
The partial amino acid sequence of the enzyme was similar to the
partial amino acid sequences of other SDR family enzymes (4,
19). This result suggests that the enzyme which we purified belongs to the SDR family. Although the NH2-terminal amino
acid sequence of levodione reductase exhibited significant similarity to the sequence of cis-benzene glycol dehydrogenase of
P. putida, the former enzyme cannot oxidize
cis-benzene glycol at a concentration of 2.0 mM. Glucose
dehydrogenases from Bacillus megaterium IAM1030 have been
shown to be SDR enzymes and also have been reported to be stabilized in
the presence of 2 M NaCl (13), and one of the glucose
dehydrogenase isozymes, GlcDH-III, has been reported to be activated by
2 M NaCl. However, the effects of other monovalent cations, such as
NH4+ and K+, and kinetic parameters
with and without NaCl have not been reported. To our knowledge, we
found the first example of an SDR family enzyme which is clearly
activated by monovalent cations.
C. aquaticum M-13 can grow on a medium containing
1,4-cyclohexanedione as the sole carbon source. However, the levodione
reductase purified in this study could not reduce 1,4-cyclohexanedione. Thus, we suggest that in C. aquaticum M-13 cells
1,4-cyclohexanedione is not degraded through a pathway initiated by a
reductase. This compound may be degraded, for example, by a ring
cleavage reaction; however, the fate of 1,4-cyclohexanedione in
C. aquaticum M-13 is still unclear. Levodione reductase
seemed to be useful for production of optically active actinol;
however, the physiological role of this enzyme remains to be clarified.
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ACKNOWLEDGMENTS |
We thank Michihiko Kataoka for analyzing the
NH2-terminal amino acid sequence.
This work was supported in part by Grants-in-Aid for Scientific
Research 6382 (to M.W) and 10356004 (to S.S.) from the Ministry of
Education, Science, Sports and Culture of Japan and by Research for the
Future Program grant JSPS-RFTF 97I00302 to S.S. from the Japan Society
for the Promotion of Science.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department
of Bioscience, Fukui Prefectural University, 4-1-1 Kenjyojima,
Matsuoka-cho, Fukui 910-1195, Japan. Phone: 81-776-61-6000. Fax:
81-776-61-6015. E-mail: masaru{at}fpu.ac.jp.
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Applied and Environmental Microbiology, October 1999, p. 4399-4403, Vol. 65, No. 10
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
