Characterization of hydroxy fatty acid dehydrogenase involved in polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum AKU 1009a

Article abstract of DOI:10.1016/j.molcatb.2015.03.020

Hydroxy fatty acid dehydrogenase, which is involved in polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum AKU 1009a, was cloned, expressed, purified, and characterized. The enzyme preferentially catalyzed NADH-dependent hydrogenation of oxo fatty acids over NAD⁺-dependent dehydrogenation of hydroxy fatty acids. In the dehydrogenation reaction, fatty acids with an internal hydroxy group such as 10-hydroxy-cis-12-octadecenoic acid, 12-hydroxy-cis-9-octadecenoic acid, and 13-hydroxy-cis-9-octadecenoic acid served as better substrates than those with α- or β-hydroxy groups such as 3-hydroxyoctadecanoic acid or 2-hydroxyeicosanoic acid. The apparent K_m value for 10-hydroxy-cis-12-octadecenoic acid (HYA) was estimated to be 38 μM with a k_cat of 7.6 × 10^-3 s^-1. The apparent K_m value for 10-oxo-cis-12-octadecenoic acid (KetoA) was estimated to be 1.8 μM with a k_cat of 5.7 × 10^-1 s^-1. In the hydrogenation reaction of KetoA, both (R)- and (S)-HYA were generated, indicating that the enzyme has low stereoselectivity. This is the first report of a dehydrogenase with a preference for fatty acids with an internal hydroxy group.

Full text of DOI:10.1016/j.molcatb.2015.03.020

Journal of Molecular Catalysis B: Enzymatic 117 (2015) 7–12
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Characterization of hydroxy fatty acid dehydrogenase involved in
polyunsaturated fatty acid saturation metabolism in Lactobacillus
plantarum AKU 1009a
Michiki Takeuchia, Shigenobu Kishinoa,b,1, Si-Bum Parkb, Nahoko Kitamuraa,
Jun Ogawaa,c,∗,1
a Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
b Laboratory of Industrial Microbiology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
c Research Unit for Physiological Chemistry, Kyoto University, Kyoto, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydroxy fatty acid dehydrogenase, which is involved in polyunsaturated fatty acid saturation metabolism
in Lactobacillus plantarum AKU 1009a, was cloned, expressed, purified, and characterized. The enzyme
preferentially catalyzed NADH-dependent hydrogenation of oxo fatty acids over NAD+-dependent dehy-
drogenation of hydroxy fatty acids. In the dehydrogenation reaction, fatty acids with an internal
hydroxy group such as 10-hydroxy-cis-12-octadecenoic acid, 12-hydroxy-cis-9-octadecenoic acid, and
13-hydroxy-cis-9-octadecenoic acid served as better substrates than those with ␣- or ␤-hydroxy groups
such as 3-hydroxyoctadecanoic acid or 2-hydroxyeicosanoic acid. The apparent Km value for 10-hydroxy-
cis-12-octadecenoic acid (HYA) was estimated to be 38 ␮M with a kcat of 7.6 × 10−3 s−1. The apparent Km
Received 2 February 2015
Received in revised form 23 March 2015
Accepted 31 March 2015
Available online 6 April 2015
Keywords:
Lactic acid bacteria
Hydroxy fatty acid
Oxo fatty acid
value for 10-oxo-cis-12-octadecenoic acid (KetoA) was estimated to be 1.8 ␮M with a kcat of 5.7 × 10−1 s−1
.
In the hydrogenation reaction of KetoA, both (R)- and (S)-HYA were generated, indicating that the enzyme
has low stereoselectivity. This is the first report of a dehydrogenase with a preference for fatty acids with
an internal hydroxy group.
Short-chain dehydrogenase/reductase
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
linoleic acid [7–10]. The novel saturation metabolism consisted
of four enzymes: CLA-HY (hydratase/dehydratase) [6,11,12], CLA-
Functional lipids have attracted attention both nutritionally and
pharmaceutically. Conjugated linoleic acid (CLA) is a representative
functional fatty acid, which has beneficial effects such as decreasing
body fat content [1] and preventing tumorigenesis [2,3] and arte-
riosclerosis [4]. Oxo fatty acids as well as CLA have also been proven
to have novel physiological functions. For example, it has recently
been reported that 13-oxo-9,11-octadecadienoic acid in tomato
juice acts as a potent peroxisome proliferator activated receptor ␣
(PPAR␣) agonist and improves dyslipidemia and hepatic steatosis
induced by obesity [5].
DH (dehydrogenase), CLA-DC (isomerase), and CLA-ER (enone
reductase) [6,12]. This saturation metabolism included some oxo
fatty acids, such as 10-oxo-cis-12-octadecenoic acid (KetoA), 10-
oxooctadecanoic acid, and 10-oxo-trans-11-octadecenoic acid, as
intermediates. These oxo fatty acids are expected to have new
physiological activities. CLA-DH generated these oxo fatty acids
through dehydrogenation of the corresponding hydroxy fatty acids,
e.g., dehydrogenation of HYA to KetoA.
In this study, we describe the enzymatic and physiochemi-
cal characteristics of CLA-DH, which is involved in the saturation
metabolism and catalyzes the dehydrogenation of hydroxy fatty
acids and the hydrogenation of oxo fatty acids.
In our previous study, we revealed polyunsaturated fatty
acid saturation metabolism in Lactobacillus plantarum AKU 1009a
[6], which is a strain with a potential to produce CLA from
2. Materials and methods
2.1. Chemicals
∗
Corresponding author at: Kyoto University, Division of Applied Life Science,
Graduate School of Agriculture, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-
8502, Japan. Tel.: +81 75 753 6115; fax: +81 75 753 6113.
HYA, 10-hydroxyoctadecanoic acid, 10-hydroxy-trans-11-
octadecenoic acid, (S)-10-hydroxy-cis-12,cis-15-octadecadienoic
acid, (S)-10-hydroxy-cis-6,cis-12-octadecadienoic acid, and
E-mail address: ogawa@kais.kyoto-u.ac.jp (J. Ogawa).
1
These corresponding authors contributed equally to this work.
http://dx.doi.org/10.1016/j.molcatb.2015.03.020
1381-1177/© 2015 Elsevier B.V. All rights reserved.

8
M. Takeuchi et al. / Journal of Molecular Catalysis B: Enzymatic 117 (2015) 7–12
13-hydroxy-cis-9-octadecenoic acid were prepared as previously
described [6,11,13,14]. Oxo fatty acids (KetoA, 10-oxooctadecanoic
acid, 10-oxo-trans-11-octadecenoic acid, 10-oxo-cis-12,cis-15-
octadecadienoic acid, 10-oxo-cis-6,cis-12-octadecadienoic acid,
12-oxo-cis-9-octadecenoic acid, and 13-oxo-cis-9-octadecenoic
acid) were prepared from hydroxy fatty acids (HYA, 10-
hydroxyoctadecanoic acid, 10-hydroxy-trans-11-octadecenoic
acid, (S)-10-hydroxy-cis-12,cis-15-octadecadienoic acid, (S)-10-
hydroxy-cis-6,cis-12-octadecadienoic acid, ricinoleic acid, and
13-hydroxy-cis-9-octadecenoic acid) by Jones oxidation, which
is oxidation of the hydroxy group with CrO3 [15]. (R)-HYA was
purified from racemic HYA using HPLC equipped with a chiral
column in the same method as “Enantiomeric purity analysis of
hydroxy fatty acids” shown in below. Fatty acid-free (<0.02%)
bovine serum albumin (BSA) was purchased from Sigma (St.
Louis, USA). All other chemicals were of analytical grade and were
commercially obtained.
CLA-DH. One unit was defined as the amount of enzyme that cat-
alyzes the conversion of 1 ␮mol of HYA per minute. The reactions
were performed under anaerobic conditions in a sealed chamber
with an O2-absorbent (Anaeropack “Kenki,” Mitsubishi Gas Chem-
ical Co., Ltd., Tokyo, Japan) and gently shaken (120 strokes/min)
at 37 ◦C for 15 min. All experiments were performed in triplicate.
Reactions were performed under the standard reaction conditions
with some modifications, as described below. The optimal reaction
temperature was determined by incubating 1 mL of the reac-
tion mixture (20 mM sodium succinate buffer, pH 4.5) at various
temperatures for 15 min under anaerobic conditions. The opti-
mal reaction pH was determined at 37 ◦C using 1 mL of 20 mM
sodium citrate buffer (pH 3.0–4.0) or 20 mM sodium succinate
buffer (pH 4.0–5.5). Thermal stability was determined by measur-
ing the enzyme activity after incubating 1 mL of reaction mixture
containing 20 mM sodium succinate buffer (pH 4.5) at various tem-
peratures for 15 min under anaerobic conditions. The pH stability
was determined by measuring enzyme activity after incubating at
37 ◦C for 10 min in the following buffers under anaerobic condi-
tions: sodium citrate buffer (50 mM; pH 3.0–4.0), sodium succinate
buffer (50 mM; pH 4.0–6.0), KPB (50 mM; pH 5.5–8.0), and Tris–HCl
buffer (50 mM; pH 7.0–9.0).
2.2. Preparation of CLA-DH
Escherichia coli Rosetta2/pCLA-DH [12] cells were cul-
tured in 1.5 L of Luria-Bertani (LB) medium at 37 ◦C for 2 h
with simultaneous shaking at 100 rpm, and then isopropyl-␤-
thiogalactopyranoside (IPTG) was added to a final concentration of
1.0 mM. After adding IPTG, the transformed cells were cultivated
at 20 ◦C for 8 h with simultaneous shaking at 100 rpm. After culti-
vation, the transformed cells (8 g) were harvested, suspended in a
standard buffer (16 mL), and treated with an ultrasonic oscillator
(5 min, 4 times, Insinator 201 M; Kubota, Japan). The standard
buffer contained 1 mM DTT and 10% (v/v) ethylene glycol in 20 mM
potassium phosphate buffer (KPB) (pH 6.5). The cell debris was
removed by centrifuging at 1700 × g for 10 min. The resulting
supernatant solutions were used as cell-free extracts. The cell-free
extracts were fractioned ultracentrifugation at 100,000 × g for
60 min and the supernatant was obtained. CLA-DH was purified
from this supernatant using a fast protein liquid chromatography
(FPLC) system (GE Healthcare) equilibrated with the standard
buffer. The supernatant was applied to a HiLoad 26/60 Superdex
200 prep-grade column (GE Healthcare) that had already been
equilibrated with standard buffer and eluted. CLA-DH was further
purified using a Mono Q 10/100 GL column (GE Healthcare), a
Superdex 200 10/300 GL column (GE Healthcare), and a Phenyl
Superose HR 10/10 (Pharmacia). The purified CLA-DH was dialyzed
with the standard buffer including 50% (v/v) glycerol and stored at
−20 ◦C until further use.
2.5. Kinetic analysis
All procedures were performed in an anaerobic chamber. Reac-
tions were performed under standard reaction conditions with
modified substrate and enzyme concentrations. The kinetics of HYA
dehydrogenation were studied using 30–1000 ␮M HYA complexed
with 0.02% (w/v) BSA as the substrate, 7 ␮g/mL CLA-DH, and a reac-
tion time of 15 min. The kinetics of KetoA dehydrogenation were
studied using 1–20 ␮M KetoA complexed with 0.02% (w/v) BSA as
the substrate, 0.35 ␮g/mL CLA-DH, and a reaction time of 5 min.
The kinetic parameters were calculated by using the experimental
data with the Michaelis–Menten equation using KaleidaGraph 4.0
(Synergy Software Inc., PA, USA).
2.6. Lipid analysis
Before lipid extraction, n-heptadecanoic acid was added
to the reaction mixture as an internal standard. Lipids were
extracted from 1 mL of the reaction mixture using 5 mL of chloro-
form/methanol/1.5% (w/v) KCl in H2O (2:2:1, by volume) according
to the procedure of Bligh–Dyer, and then concentrated by evap-
oration under reduced pressure [16]. The resulting lipids were
dissolved in 5 mL of benzene/methanol (3:2, by volume) and
methylated with 300 ␮L of 1% trimethylsilyldiazomethane (in hex-
ane) at 28 ◦C for 30 min. After methyl esterification, the resulting
fatty acid methyl esters were concentrated by evaporation under
reduced pressure. The resulting fatty acid methyl esters were ana-
lyzed by gas–liquid chromatography (GC) using a Shimadzu (Kyoto,
Japan) GC-1700 gas chromatograph equipped with a flame ion-
ization detector, a split injection system, and a capillary column
(SPB-1, 30 m × 0.25 mm I.D., SUPELCO, PA, USA). The initial col-
umn temperature 180 ◦C (for 30 min) was subsequently increased
to 210 ◦C at a rate of 60 ◦C/min, and then maintained at 210 ◦C for
29.5 min. The injector and detector were operated at 250 ◦C. Helium
was used as a carrier gas at a flow rate of 1.4 mL/min. The fatty acid
peaks were identified by comparing the retention times to those of
known standards.
2.3. Determination of the molecular mass of CLA-DH
In order to determine the native molecular mass of CLA-
DH, the enzyme solution was subjected to high perfor-
mance gel-permeation chromatography on a G-3000SW column
(0.75 × 60 cm, Tosoh, Tokyo, Japan) at room temperature. It was
eluted with 100 mM KPB (pH 6.5) containing 100 mM Na2SO4 at
a flow rate of 0.5 mL/min. The absorbance of the effluent was
monitored at 280 nm. The molecular mass of the enzymes was
determined from their mobility relative to those of standard pro-
teins.
2.4. Reaction conditions
All operations were performed in an anaerobic chamber. The
standard reaction conditions were as described. The reactions were
performed in test tubes (16.5 × 125 mm) that contained 1 mL of
reaction mixture (20 mM sodium succinate buffer, pH 4.5) with
0.1% (w/v) HYA or KetoA complexed with BSA [0.02% (w/v)] as the
substrate, 5 mM NAD+ or NADH and 42 ␮g (=0.04 U/mL) purified
2.7. Enantiomeric purity analysis of hydroxy fatty acids
The enantiomeric purity of HYA, which was produced from
KetoA hydrogenation with CLA-DH, was analyzed using HPLC (Shi-
madzu, Kyoto, Japan) using a Shimadzu LC 20A System (Shimadzu)

M. Takeuchi et al. / Journal of Molecular Catalysis B: Enzymatic 117 (2015) 7–12
9
Table 1
Substrate specificity of CLA-DH for dehydrogenation.
Substrate
Relative activity [%]
(S)-10-Hydroxy-cis-12-octadecenoic acid (HYA)
(R)-10-Hydroxy-cis-12-octadecenoic acid ((R)-HYA)
10-Hydroxyoctadecanoic acid
100a
98
25
10-Hydroxy-trans-11-octadecenoic acid
(S)-10-Hydroxy-cis-12,cis-15-octadecadienoic acid
(S)-10-Hydroxy-cis-6,cis-12-octadecadienoic acid
(R)-12-Hydroxy-cis-9-octadecenoic acid
13-Hydroxy-cis-9-octadecenoic acid
3-Hydroxyoctadecanoic acid (C18)
69
75
54
62
142
b
–
3-Hydroxytetradecanoic acid (C14)
2-Hydroxyeicosanoic acid (C20)
–
–
Fig. 1. SDS–PAGE analysis of purified CLA-DH. Molecular mass standards: from the
top, phosphorylase b (97,200), bovine serum albumin (66,400), ovalbumin (45,000),
carbonic anhydrase (29,000), and trypsin inhibitor (20,100). The observed molecular
weight of purified CLA-DH was 40 kDa.
Methyl (S)-10-Hydroxy-cis-12-octadecenoate
8-Hexadecanol
127
24
a
The activity of (S)-10-hydroxy-cis-12-octadecenoic acid dehydrogenation
(=0.048 U/mg) under the condition (5 mM NAD+; 37 ◦C, pH 4.5, 15 min) was defined
as 100%.
b
–, Not detected.
equipped with a chiral column (Chiralpak IA, 4.6 mm I.D., Daicel,
Osaka, Japan) and an Evaporative Light Scattering Detector System
(Shimadzu, Kyoto, Japan) as a detector. Acetonitrile/0.2% formic
acid (65:35) was used as a solvent at a flow rate of 1.0 mL/min.
Racemic HYA prepared from KetoA reduction with NaBH4 [17] was
used as the standard.
Table 2
Substrate specificity of CLA-DH for hydrogenation.
Substrate
Relative activity [%]
10-Oxo-cis-12-octadecenoic acid (KetoA)
10-Oxooctadecanoic acid
100a
66
10-Oxo-trans-11-octadecenoic acid
10-Oxo-cis-12,cis-15-octadecadienoic acid
10-Oxo-cis-6,cis-12-octadecadienoic acid
12-Oxo-cis-9-octadecenoic acid
13-Oxo-cis-9-octadecenoic acid
Methyl 10-oxo-cis-12-octadecenoate
7-Hexadecanone
53
44
6
87
156
170
332
3. Results
3.1. Purification of CLA-DH
The recombinant CLA-DH without the tag was purified to homo-
geneity from cell-free extracts of the transformed E. coli through
four steps of column chromatography. The purified CLA-DH dis-
played a single band on an SDS–PAGE gel (Fig. 1). The observed
molecular mass of the subunit was 40 kDa, corresponding to a cal-
culated mass of 32 kDa deduced from the amino acid sequence of
its gene. The relative native molecular mass was estimated to be
32 kDa by HPLC on a G-3000SW column, indicating that the enzyme
consists of the single subunit. The purified CLA-DH was used for
further characterization.
a
The activity of 10-oxo-cis-12-octadecenoic acid hydrogenation (=0.22 U/mg)
under the condition (5 mM NADH; 37 ◦C, pH 4.5, 15 min) was defined as 100%.
acid, 10-hydroxy-trans-11-octadecenoic acid, (S)-10-hydroxy-
cis-12,cis-15-octadecadienoic acid, (S)-10-hydroxy-cis-6,cis-12-
octadecadienoic acid, (R)-12-hydroxy-cis-9-octadecenoic acid, and
13-hydroxy-cis-9-octadecenoic acid served as good substrates
and transformed into corresponding 10-, 12-, or 13-oxo fatty
acids. In addition, HYA methyl ester and 8-hexadecanol were
dehydrogenated to KetoA methyl ester and 8-hexadecanone,
respectively. In contrast, 2- or 3-hydroxy fatty acids such as
3-hydroxyoctadecanoic acid, 3-hydroxytetradecanoic acid, and 2-
hydroxyeicosanoic acid were not dehydrogenated (Table 1).
In the hydrogenation reaction, 10-, 12- or 13-oxo C18 fatty
acids such as KetoA, 10-oxooctadecanoic acid, 10-oxo-trans-11-
octadecenoic acid, 10-oxo-cis-12,cis-15-octadecadienoic acid, 10-
oxo-cis-6,cis-12-octadecadienoic acid, 12-oxo-cis-9-octadecenoic
acid, and 13-oxo-cis-9-octadecenoic acid served as good substrates
and transformed into corresponding 10-, 12- or 13-hydroxy fatty
acids. In addition, KetoA methyl ester and 7-hexadecanone were
hydrogenated to HYA methyl ester and 7-hexadecanol, respectively
(Table 2).
3.2. Effects of reaction conditions
CLA-DH required NAD+/NADH as
a
cofactor but not
NADP+/NADPH. The effects of NAD+/NADH concentration were
examined from 0 to 7.5 mM (Fig. 2a). The dehydrogenation and
hydrogenation activities increased with increasing concentrations
of NAD+/NADH. The effects of temperature were also examined.
The optimal reaction temperature was found to be 52 ◦C (Fig. 2b).
The effects of pH were examined over a pH range from 3.0 to 5.5
with an optimal reaction pH determined to be pH 4.5 (Fig. 2c).
3.3. Enzyme stability
The thermal stability of the purified enzyme was investigated
from 18 ◦C to 67 ◦C. The enzyme was incubated at each temperature
for 15 min at pH 4.5. More than 80% of the initial activity remained
at temperatures up to 28 ◦C (Fig. 3a). The pH stability of the purified
enzyme was investigated by incubating the enzyme in different
buffers within a pH range of 3.0 to 9.0 for 10 min at 37 ◦C. More
than 80% of the initial activity remained in a pH range from 4.5 to
7.5 (Fig. 3b).
3.5. Kinetic analysis of the CLA-DH catalyzing reactions
The substrate concentration-reaction velocity curves for HYA
dehydrogenation and KetoA hydrogenation were used with the
Michaelis–Menten equation. The apparent Km value for HYA in
the dehydrogenation reaction was estimated to be 38 ␮M with
a kcat of 7.6 × 10−3 s−1. The apparent Km value for KetoA in the
hydrogenation reaction was estimated to be 1.8 ␮M with a kcat of
3.4. Substrate specificity
In the dehydrogenation reaction, 10-, 12-, or 13-hydroxy
C18 fatty acids such as HYA, (R)-HYA, 10-hydroxyoctadecanoid
5.7 × 10−1 s−1
.

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M. Takeuchi et al. / Journal of Molecular Catalysis B: Enzymatic 117 (2015) 7–12
Fig. 2. Effects of NAD+/NADH concentrations, temperature, and pH on the activity of CLA-DH. (a) Effects of NAD+/NADH concentrations: dehydrogenation activity (closed
circles) and hydrogenation activity (open circles) were assayed under standard reaction conditions, except for NAD+/NADH concentrations. (b) Effects of temperature:
dehydrogenation activity (closed circles) and hydrogenation activity (open circles) were assayed under standard reaction conditions, except for the temperature. (c) Effects of
pH: activity was assayed under standard reaction conditions, except for the buffers used. Sodium citrate buffer (closed and open circles for dehydrogenation and hydrogenation,
respectively), pH 3.0–4.0, and sodium succinate buffer (closed and open triangles for dehydrogenation and hydrogenation, respectively), pH 4.0–5.5, were used.
3.6. Enantiomeric purities of the hydroxy fatty acids produced by
CLA-DH
fatty acid saturation metabolism in L. plantarum AKU 1009a [6].
These results suggested that CLA-DH plays an important role in
saturation metabolism.
The enantiomeric purity of HYA produced from KetoA
by CLA-DH was analyzed using HPLC with a chiral column.
Almost the same amounts of both enantiomers of (R)-HYA
and (S)-HYA were produced from KetoA, indicating that CLA-
DH had low stereoselectivity in oxo fatty acid hydrogena-
tion.
CLA-DH, which belongs to the short-chain dehydroge-
nase/reductase (SDR) family, showed considerable similarity
with other SDRs (Fig. 4). In this paper, we characterized CLA-DH
from the aspect of its physiological function to clarify its distinct
characteristic properties in the SDR family, especially from the
viewpoint of substrate specificity. There are few reports regarding
either hydroxy fatty acid dehydrogenation or oxo fatty acid
hydrogenation in the SDR family. However, CLA-DH catalyzed the
dehydrogenation or hydrogenation of fatty acids which have an
internal hydroxy or an oxo group, respectively (Tables 1 and 2).
Micrococcus luteus WIUJH-20 was reported to convert 10- or 12-
hydroxyoctadecanoic acid to the corresponding oxooctadecanoic
acid. The amino acid sequence of the enzyme which catalyzes
the above oxidation of hydroxy fatty acid in M. luteus WIUJH-20
belongs to a secondary alcohol dehydrogenase [18]. The amino
acid sequence of the secondary alcohol dehydrogenase from
M. luteus did not resemble that of CLA-DH, indicating that the
dehydrogenation activity of CLA-DH was characteristic activity
among the SDR family.
3.7. Effects of chemicals on the enzyme activity
The effects of metal ions and inhibitors (1 mM) were inves-
tigated in both the hydration and dehydration reactions. The
reactions were strongly inhibited by Ag+, Cu2+, Hg2+, VO3−, WO4
,
2−
and aluminon (data not shown). 2,3,5-Triphenyltetrazolium
inhibited only dehydrogenation activity.
4. Discussion
We revealed polyunsaturated fatty acid saturation metabolism
in L. plantarum AKU 1009a and identified CLA-DH. The CLA-DH gene
was located together with CLA-DC (fatty acid isomerase) and CLA-
ER (fatty acid enone reductase) genes involved in polyunsaturated
As a characteristic property of CLA-DH, the enzyme showed
higher activity in hydrogenation than dehydrogenation reactions.
Fig. 3. Effects of temperature and pH on stability of CLA-DH. (a) Effect of temperature: the thermal stability of the dehydrogenation activity (closed circles) and hydrogenation
activity (open circles) were assessed under standard reaction conditions after incubation at each temperature (18–67 ◦C) for 30 min. The activities after incubation at 18 ◦
C
were defined as 100% for dehydrogenation (0.048 U/mg) and hydrogenation (0.22 U/mg). (b) Effect of pH: the pH stabilities of the dehydrogenation (closed) and hydrogenation
(open) reactions were evaluated under standard reaction conditions after incubation at 37 ◦C for 10 min at each pH. Sodium citrate buffer, pH 3.0–4.0 (circles), sodium succinate
buffer, pH 4.0–6.0 (triangles), potassium phosphate buffer, pH 5.0–7.5 (diamonds), and Tris–HCl buffer, 7.0–9.0 (squares) were used. The activities after incubation in sodium
succinate buffer (pH 4.5) were defined as 100% for dehydrogenation (0.042 U/mg) and hydrogenation (0.22 U/mg).

M. Takeuchi et al. / Journal of Molecular Catalysis B: Enzymatic 117 (2015) 7–12
11
Fig. 4. Multiple-sequence alignment of CLA-DH and ADHs belonging to the SDR family. The SDR family includes Rhodococcus erythropolis (AADH), Lactobacillus brevis (LbRADH),
Thermus thermophilus (TtADH), and Leifsonia sp. strain S749 (LSADH). The accession numbers of the listed proteins are as follows: CLA-DH, BAL42247; AADH, BAF43657;
LbRADH, YP 794544; TtADH, YP 003977; LSADH, BAD99642. Black and gray shading indicate residues highly conserved in the SDR family. TGXXXGXG is co-enzyme binding
region in typical SDRs. The star indicates the four members of catalytic tetrad.
The activity of KetoA hydrogenation was 5 times higher than that
of HYA dehydrogenation (Fig. 2).
perspective, CLA-DH is useful for the production of oxo fatty acids
with unique physiological functions in combination with fatty acid
hydratases such as CLA-HY (11, 13), which were reported as good
catalysts generating hydroxy fatty acids from common C18 fatty
acids.
Although many SDRs have high enantioselectivity [19–23], CLA-
DH had low enantioselectivity to dehydrogenate both (R) and (S)
hydroxy fatty acids (Table 1) and produce (R) and (S) hydroxy fatty
acids from oxo fatty acid. In addition, CLA-DH dehydrogenated 10-,
12-, and 13-hydroxy fatty acids (Table 1), indicating its low regio-
selectivity.
Acknowledgments
In our previous study, we reported the production of many
kinds of hydroxy fatty acids such as 10- and 13-hydroxy octade-
capolyenoic acid [6,11,13,14]. Using these various hydroxy fatty
acids and hydroxy fatty acid dehydrogenase such as CLA-DH, we
can provide many kinds of corresponding oxo fatty acids by apply-
ing the wide substrate specificity of CLA-DH. These results enable
us to provide new functional lipids, oxo fatty acids.
This work was supported, in part, by the Industrial Technol-
ogy Research Grant Program in 2007 (Grant 07A08005a to S.K.);
the Project for the Development of a Technological Infrastruc-
ture for Industrial Bioprocesses on Research and Development of
New Industrial Science and Technology Frontiers (S.S.) from the
New Energy and Industrial Technology Development Organiza-
tion (NEDO) of Japan; Scientific Research Grants in Aid 19780056
(to S.K.), 16688004 (to J.O.), and 18208009 (to S.S.); the Cen-
ters of Excellence for Microbial-Process Development Pioneering
Future Production Systems from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (J13 to S.S.); the Bio-
Oriented Technology Research Advancement Institution of Japan
(J.O.); Science and Technology Promotion Program for Agriculture
Forestry, Fisheries and Food Industry from the Ministry of Agricul-
ture, Forestry and Fisheries of Japan (26002A to J.O.); Advanced Low
Carbon Technology Research and Development Program of Japan
5. Conclusions
The properties of CLA-HY, a novel hydroxy fatty acid dehydro-
genase from L. plantarum were investigated. CLA-DH showed wide
substrate specificity toward hydroxy fatty acids with a preference
to those with an internal hydroxy group. Such substrate prefer-
ence explained well that CLA-DH is involved in polyunsaturated
fatty acid saturation metabolism. From an application oriented

12
M. Takeuchi et al. / Journal of Molecular Catalysis B: Enzymatic 117 (2015) 7–12
(S.K.); M.T. (26686) and S.K. (1401985) received Research Fellow-
ship from the Japan Society for the Promotion of Science for Young
Scientists.
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