Properties and tissue distribution of a novel aldo-keto reductase encoding in a rat gene (Akr1b10)

Article abstract of DOI:10.1016/j.abb.2010.08.010

A recent rat genomic sequencing predicts a gene Akr1b10 that encodes a protein with 83% sequence similarity to human aldo-keto reductase (AKR) 1B10. In this study, we isolated the cDNA for the rat AKR1B10 (R1B10) from rat brain, and examined the enzymatic properties of the recombinant protein. R1B10 utilized NADPH as the preferable coenzyme, and reduced various aldehydes (including cytotoxic 4-hydroxy-2-hexenal and 4-hydroxy- and 4-oxo-2-nonenals) and α-dicarbonyl compounds (such as methylglyoxal and 3-deoxyglucosone), showing low K_m values of 0.8-6.1μM and 3.7-67μM, respectively. The enzyme also reduced glyceraldehyde and tetroses (K_m=96-390μM), although hexoses and pentoses were inactive and poor substrates, respectively. Among the substrates, 4-oxo-2-nonenal was most efficiently reduced into 4-oxo-2-nonenol, and its cytotoxicity against bovine endothelial cells was decreased by the overexpression of R1B10. R1B10 showed low sensitivity to aldose reductase inhibitors, and was activated to approximately two folds by valproic acid, and alicyclic and aromatic carboxylic acids. The mRNA for R1B10 was expressed highly in rat brain and heart, and at low levels in other rat tissues and skin fibroblasts. The results suggest that R1B10 functions as a defense system against oxidative stress and glycation in rat tissues.

Full text of DOI:10.1016/j.abb.2010.08.010

Archives of Biochemistry and Biophysics 503 (2010) 230–237
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
Properties and tissue distribution of a novel aldo–keto reductase encoding
in a rat gene (Akr1b10)
a
a
c
b
d
Satoshi Endo a,b, , Toshiyuki Matsunaga , Tsukasa Kuragano , Satoshi Ohno , Yukio Kitade , Kazuo Tajima ,
*
Ossama El-Kabbani e, Akira Hara a
a Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan
b United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1193, Japan
c Faculty of Engineering, Gifu University, Gifu 501-1193, Japan
d Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181, Japan
e Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Vic. 3052, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 June 2010
and in revised form 7 August 2010
Available online 13 August 2010
A recent rat genomic sequencing predicts a gene Akr1b10 that encodes a protein with 83% sequence sim-
ilarity to human aldo–keto reductase (AKR) 1B10. In this study, we isolated the cDNA for the rat AKR1B10
(R1B10) from rat brain, and examined the enzymatic properties of the recombinant protein. R1B10 uti-
lized NADPH as the preferable coenzyme, and reduced various aldehydes (including cytotoxic 4-
hydroxy-2-hexenal and 4-hydroxy- and 4-oxo-2-nonenals) and
ylglyoxal and 3-deoxyglucosone), showing low Km values of 0.8–6.1
enzyme also reduced glyceraldehyde and tetroses (Km = 96–390
a
-dicarbonyl compounds (such as meth-
M and 3.7–67 M, respectively. The
M), although hexoses and pentoses
Keywords:
Aldo–keto reductase superfamily
AKR1B10
l
l
l
were inactive and poor substrates, respectively. Among the substrates, 4-oxo-2-nonenal was most effi-
ciently reduced into 4-oxo-2-nonenol, and its cytotoxicity against bovine endothelial cells was decreased
by the overexpression of R1B10. R1B10 showed low sensitivity to aldose reductase inhibitors, and was
activated to approximately two folds by valproic acid, and alicyclic and aromatic carboxylic acids. The
mRNA for R1B10 was expressed highly in rat brain and heart, and at low levels in other rat tissues and
skin fibroblasts. The results suggest that R1B10 functions as a defense system against oxidative stress
and glycation in rat tissues.
Aldose reductase
Glycation
Oxidative stress
4-Oxo-2-nonenal
4-Hydroxy-2-nonenal
3-Deoxyglucosone
Ó 2010 Elsevier Inc. All rights reserved.
Introduction
dins [11]. In humans, a NADPH-dependent AKR that shares high
overall amino acid sequence identity (71%) with AR (AKR1B1),
Aldose reductase (AR)1 catalyzes the first step in the polyol
pathway and is considered to be one of the most extensively stud-
ied members of the aldo–keto reductase (AKR) superfamily [1], be-
cause of its potential roles in mediating hyperglycemic injury and
the development of secondary diabetic complications [2–4]. In
addition to polyol synthesis, the enzyme shows broad substrate
specificity for carbonyl compounds of physiological significance,
including lipid peroxidation products, such as 4-hydroxy-2-none-
nal (HNE) [5] and 4-oxo-2-nonenal (ONE) [6], advanced glycation
end-product (AGE) precursors (methylglyoxal and 3-deoxygluco-
sone [7,8]), isocaproaldehyde [9], retinoids [10], and prostaglan-
was recently identified [12,13], and is named AKR1B10 in the
AKR superfamily. AKR1B10 shows similar broad substrate specific-
ity to human AR, but clearly differs from AR in its efficient reduc-
tion of retinals, isoprenyl aldehydes and isatin, inability to reduce
aldohexoses [10,14], and high susceptibility to inhibition by bile
acids [14]. AKR1B10 is ubiquitously distributed in human tissues
[12,13], and up-regulated in lung and hepatic carcinomas (squa-
mous cell carcinoma and adenocarcinoma) [12,15], indicating its
role as a tumor marker.
Recent genomic analyses have shown that each three genes for
AR-like proteins are clustered on mouse chromosome 6B1 and rat
chromosome 4q22 (Entrez Gene: www.ncbi.nlm.nih.gov/gene).
Among the proteins, each two proteins (AKR1B7 and AKR1B8 in
mice, and AKR1B13 and AKR1B14 in rats) have been studied in
terms of the enzymatic properties, tissue distribution and gene
regulation. Mouse AKR1B8 and rat AKR1B13 show similar broad
substrate specificity for various aldehydes and are inhibited by
AR inhibitors [16,17], whereas mouse AKR1B7 and rat AKR1B14 ex-
hibit prostaglandin (PG) F synthase activity [11,18] in addition to
* Corresponding author at: Laboratory of Biochemistry, Gifu Pharmaceutical
University, Gifu 501-1196, Japan. Fax: +81 58 230 8105.
E-mail address: sendo@gifu-pu.ac.jp (S. Endo).
1
Abbreviations used: AGE, advanced glycation end-product; AKR, aldo–keto reduc-
tase; AR, aldose reductase; DHN, 1,4-dihydroxy-2-nonene; HNE, 4-hydroxy-2-none-
nal; R1B10, rat AKR1B10; ONE, 4-oxo-2-nonenal; ONOL, 4-oxo-2-nonenol; RT, reverse
transcription.
0003-9861/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2010.08.010

S. Endo et al. / Archives of Biochemistry and Biophysics 503 (2010) 230–237
231
reductase activity for several aldehydes such as isocaproaldehyde
and HNE [19–21]. AKR1B8 and AKR1B13 are sensitive to AR inhib-
itors [16,17], which do not inhibit mouse AKR1B7 [20]. Like human
AKR1B10, AKR1B13 is almost ubiquitously expressed in rat tissues
[17], whereas the other rodent enzymes show tissue-specific
expression [19–22]. These findings suggest that in rodents the four
AR-like proteins play tissue-specific roles. For AKR1B7 and
AKR1B14 that are highly expressed in mouse and rat adrenal
glands, their PGF synthase activity is suggested to regulate the
adrenal catecholamine release [18]. Currently, the uncharacterized
rodent AR-like proteins are products of the third mouse and rat
genes, both of which are tentatively named Akr1b10. The two
316-amino acid proteins predicted from the genes share 92% se-
quence identity and exhibit 83% sequence identity with human
AKR1B10, but their biochemical properties have yet to be explored.
In this study, we have isolated the cDNA for rat AKR1B10-like
protein (R1B10) from the brain, and examined the enzymatic prop-
erties of the recombinant R1B10, tissue distribution and gene reg-
ulation. The data indicated that R1B10 acts as a reductase for
reactive carbonyl compounds derived from lipid peroxidation
and glycation mainly in rat brain and heart.
tract was prepared as described previously [28]. The recombinant
R1B10 was purified at 4 °C by subsequent three column chroma-
tography steps in 10 mM Tris–HCl buffer, pH 8.0, containing
5 mM 2-mercaptoethanol, 0.5 mM EDTA and 20% (v/v) glycerol to
stabilize the enzyme. The E. coli cell extract was dialyzed against
the Tris–HCl buffer and applied to
a Q-Sepharose column
(2 cm ꢀ 40 cm). The enzyme did not adsorb on the column, and
the enzyme fractions were concentrated by ultrafiltration, and
gel-filtrated through a Sephadex G-100 column (2.5 cm ꢀ 90 cm).
The enzyme fractions were applied to a Red-Sepharose column
(1.5 ꢀ 5 cm), and the enzyme was eluted with the buffer contain-
ing 0.5 mM NADP+ after the column was washed with the buffer
without NADP+. The identity of the purified recombinant protein
with R1B10 encoded in the cDNA was verified by the mass spec-
trometry analysis of its 10 lysylendopeptidase-digested peptides
using a Bruker-Franzen Mass Spectrometry System Ultraflex TOF/
TOF [28]. The concentration of the purified R1B10 was determined
by UV absorption using e
280 = 71,112 Mꢁ1 cmꢁ1, which was deter-
mined by a dry weight measurement of the lyophilized enzyme,
assuming a molecular weight of 38,028, because the recombinant
enzyme comprising of a sequence of a six-His tag and ten amino
acids prior to its N-terminal Met.
Materials and methods
Assay of enzyme activity
Materials
The reductase activity of AKRs was determined by measuring
the rate of change in NADPH absorbance at 340 nm. The standard
reaction mixture consisted of 0.1 M potassium phosphate, pH 7.4,
0.1 mM NADPH, 0.1 mM pyridine-3-aldehyde and enzyme in a to-
tal volume of 2.0 mL. The dehydrogenase activity was assayed by
measuring change in NADPH fluorescence (at 455 nm with an exci-
tation wavelength of 340 nm) in the phosphate buffer, pH 7.4, con-
taining 0.25 mM NADP+ and an appropriate amount of alcohol
substrate. The reductase activity for all-trans-retinal was measured
by the method of Parés and Julià [29], except that 0.1 M potassium
phosphate, pH 7.4, containing 0.01% Tween 80 was used as the buf-
fer. The substrates, which are hardly soluble in water, were dis-
solved in methanol or 50% methanol, and added into the reaction
mixture, in which the final concentration of methanol was less
than 1.2%. The concentration of methanol did not affect the activity
of the enzymes. One unit of enzyme activity was defined as the
amount of enzyme that catalyzes the oxidation or formation of
ONE, 4-hydroxy-2-hexenal and PGs were from Cayman, resins
for column chromatography and an enhanced chemiluminescence
substrate system were from Amersham Biosciences; and a pCold I
expression vector and Taq DNA polymerase were from Takara
(Kusatsu, Japan). Pfu DNA polymerase and Escherichia coli BL21
(DE3) pLysS were purchased from Stratagene and Invitrogen,
respectively. Rat glioma cell line, C6, was obtained from American
Type Culture Collection (Manassas, VA), and bovine aortic endothe-
lial cells (BAECs) were a generous gift from Taisho Pharmaceutical
Co. (Saitama, Japan). Isocaproaldehyde [9], HNE [23], 21-dehydro-
cortisol [24], trans-1,2-dihydroxy-1,2-dihydrobenzene (benzene
dihydrodiol) [25] and trans-1,2-dihydroxy-1,2-dihydronaphtha-
lene (naphthalene dihydrodiol) [26] were synthesized as described
previously. 1,4-Dihydroxy-2-nonenal (DHN), 4-oxo-2-nonenol
(ONOL) and a glutathione adduct of ONE (GS-ONE) were prepared
by the methods of Doorn et al. [6]. All other chemicals were of the
highest grade that could be obtained commercially.
1
lmol NADPH per minute at 25 °C.
The apparent Km and kcat values were determined over a range
of five or six substrate concentrations at a saturating concentration
of coenzyme by fitting the initial velocities to the Michaelis–Men-
ten equation. The kinetic study in the presence of an inhibitor was
performed in a similar manner, and the inhibitor constant (Ki) of
the competitive inhibitor with respect to 1-octanol in the reverse
reaction was calculated from Dixon plot. The initial velocity analy-
cDNA isolation
The cDNA for R1B10 was isolated from the total RNA prepara-
tion of rat brain by reverse transcription (RT)-PCR. The RT and
DNA techniques followed the standard procedures described by
Sambrook et al. [27]. PCR was performed with Pfu DNA polymerase
and a pair of sense and antisense primers, 50-ttttcatatggccgccttcg-
30, and 50-ccccggatcctcagtattc-30, which contain underlined NdeI
and BamHI sites, respectively. The PCR products were purified, di-
gested with the two restriction enzymes, and ligated into the pCold
I vectors that had been digested with the two restriction enzymes.
The expression constructs were transfected into E. coli BL21 (DE3)
pLysS cells. The insert of the cloned cDNA was sequenced by using
a Beckman CEQ8000 DNA sequencer.
sis for determining the dissociation constant (KA),
a and b values
for the activator was carried out with six concentrations of pyri-
dine-3-aldehyde and NADPH as the varied and fixed substrates,
respectively. The kinetic values were calculated from secondary re-
plots of a 1/D slope or 1/D intercept versus 1/[activator] [30]. The
kinetic constants were expressed as the means of at least two
determinations.
Identification of reduced product of ONE and HNE
Expression and purification of recombinant enzyme
The product identification was carried out with following minor
modifications as described by Doorn et al. [6]. Reaction was con-
The E. coli cells were cultured in a LB medium (1 L) containing
ducted in a 10 mL system containing 0.1 mM NADPH, 10
lM ONE
ampicillin (50
reached 0.5. Isopropyl-b-
added and the culture was continued for 24 h at 15 °C. The cell ex-
l
g/mL) at 37 °C until the absorbance at 600 nm
or HNE, R1B10 (25 g) and 0.1 M potassium phosphate, pH 7.4.
The substrate and products were extracted into 20 mL ethyl ace-
tate 30 min after the reaction was started at 25 °C, and analyzed
l
D
-thiogalactopyranoside (0.1 mM) was

232
S. Endo et al. / Archives of Biochemistry and Biophysics 503 (2010) 230–237
by TLC. The compounds and standards (ONE, ONOL and DHN) were
visualized by spraying with phosphomolybdic acid (10% in etha-
nol) and heating at 105 °C for 5 min.
firmed to be identical to that of the mRNA for R1B10 (Accession
No. NM_001013084) predicted from the rat genomic analysis.
The protein encoded in the cDNA was expressed in E. coli cells,
and detected as a 37-kDa protein on SDS–PAGE analysis of the cell
extract. The recombinant R1B10 was purified to homogeneity by
detecting the 37-kDa protein on the SDS–PAGE analysis. The se-
quences (composed of a total 197 residues) of 10 lysylendopepti-
dase-digested peptides derived from the purified enzyme were
identical to those deduced from the cDNA for R1B10. The enzyme
was eluted at a low molecular weight of approximately 35 kDa on
the Sephadex G-100 column chromatography step, suggesting its
monomeric nature.
Tissue distribution analysis
Tissues were excised from 12 to 14-week-old male and female
Wistar rats, and their total RNAs and cytosolic fractions were pre-
pared as described [27,28]. The total RNA samples were subjected
to RT-PCR using Taq DNA polymerase. In the PCR of the cDNA for
R1B10, a pair of sense and antisense primers, (50-atggccgccttcgt-
gaag-30 and 50-aattcagcttcagtattccga-30, respectively) were used,
and the amplification consisted of an initial denaturation step at
94 °C for 5 min, and 30 cycles of denaturation (94 °C/30 s), anneal-
ing (57 °C/30 s) and extension (72 °C/1 min), and a final incubation
step at 72 °C for 5 min. The PCR amplification of the cDNAs for
AKRs (1B4, 1B13 and 1B14) was performed as described previously
[17]. The cDNA for rat b-actin was also amplified as an internal
control using the specific primers that were obtained from Toyobo
(Osaka, Japan). The PCR products were separated by agarose gel
electrophoresis, and revealed with ethidium bromide. The cyto-
solic fractions of the tissue homogenates were subjected to Wes-
tern blot analysis using polyclonal antibodies against human
AKR1B10, which were raised in rabbits [31]. The immunoreactive
proteins were visualized using the enhanced chemiluminescence
substrate system.
Substrate specificity of R1B10
The purified R1B10 exhibited NADPH-linked reductase activity
(0.11 U/mg) towards 0.1 mM pyridine-3-aldehyde at pH 7.4, and
Km and kcat values for NADPH were 1.0
tively. The enzyme also utilized NADH as the coenzyme, but the Km
and kcat values for this coenzyme were 0.15 mM and 1.3 minꢁ1
l
M and 4.1 minꢁ1, respec-
,
respectively. Therefore, the substrate specificity of R1B10 was
examined using the preferable coenzyme NADPH.
The enzyme efficiently reduced aromatic aldehydes (Table 1),
although the Km values for furfural and its 5-hydroxymethyl deriv-
ative were slightly high. Aliphatic aldehydes, except for 2- and 3-
carbon aldehydes (glycolaldehyde and acrolein), were excellent
substrates with Km values in low
lM range, and substrate inhibi-
tion was observed at more than 10-fold concentrations of their
Km values. When the catalytic efficiency was compared between
the alkanal and alkenal with the same chain length, alkanals were
reduced more efficiently than alkenals. Among the alkenals, the
kcat/Km values for ONE, HNE and 4-hydroxy-2-hexenal were more
than 2-fold higher than those for the corresponding 1-nonenal
and 1-hexenal, indicating that the introduction of the oxo or hy-
droxyl group at position 4 of the alkenals increases the catalytic
efficiency. All-trans-retinal and farnesal, excellent substrates for
human AKR1B10 [10,17], were less efficiently reduced by R1B10.
The reduction products of ONE and HNE were identified as their
alcohols, ONOL and DHN, respectively, by TLC analysis. R1B10 also
reduced the glutathione adduct of ONE (GS-ONE) with a 10-fold
Cell culture experiments
Rat skin fibroblasts and coronary aortic endothelial cells were
isolated and cultured according to the method of Domenighetti
et al. [32] with minor modification [33]. C6 cells and BAECs were
cultured in Dulbecco’s modified Eagle medium supplemented with
10% fetal bovine serum, penicillin (100 U/mL) and streptomycin
(100 lg/mL) at 37 °C in a humidified incubator containing 5%
CO2. Total RNAs were isolated from the cells and analyzed for the
expression of the mRNA for R1B10 by RT-PCR as described above.
The transfection of the pGW1 plasmids harboring the cDNA for
R1B10 into BAECs was performed as described previously [17].
The cDNA for R1B10 was initially amplified from the above bacte-
rial expression vector by PCR using the following primers. The
sense primer, 50-ccccgaattcGCCACCatggccgccttcgt-30, contains an
EcoRI site, a Kozak sequence and a start codon, which are shown
in italic, capital and underlined letters, respectively. The antisense
primer, 50-ccccgtcgactcagtattccga-30, is complementary to the bac-
terial expression vector containing a SalI site, which is shown in
italic letters. After the sequences of the PCR products were verified,
they were subcloned at the EcoRI and SalI sites of the pGW1 vector.
Using a Lipofectamine 2000 Reagent (Invitrogen), the cells were
transfected for 48 h with the expression plasmids. The transfected
cells were maintained in the medium containing 2% fetal bovine
serum for 24 h, and then used for the experiments. The overexpres-
sion of R1B10 in the cell extract was measured by Western blotting
using the antibodies against human AKR1B10. Following the treat-
ment of ONE or HNE for 24 h, the cell viability was evaluated by a
tetrazolium dye-based cytotoxicity assay, and expressed as % of
control culture conditions as described previously [34]. All cell
incubations were performed in triplicate.
Table 1
Kinetic constants for aldehydes.
Substrate
Km
(l
M) kcat (minꢁ1
)
kcat/Km (minꢁ1 Mꢁ1
l )
Aromatic aldehydes
4-Nitrobenzaldehyde
Pyridine-3-aldehyde
Benzaldehyde
0.7
2.5
9.1
3.0
4.1
3.2
1.5
2.5
4.3
1.6
0.35
0.083
0.018
Furfural
18
5-Hydroxymethylfurfural 138
Alkanals
1-Octanal
1-Decanal
1-Nonanal
1-Hexanal
Isocaproaldehyde
Glycolaldehyde
0.8
1.7
1.5
2.1
11
720
4.2
7.3
4.6
2.6
1.7
2.7
5.3
4.3
3.1
1.2
0.15
0.004
Alkenals
ONE
HNE
0.8
6.2
3.4
2.1
1.0
2.2
3.7
3.4
0.94
0.58
2.0
2.9
7.8
2.1
1.3
1.1
0.85
0.60
0.39
0.26
0.22
0.15
0.059
1.6
1.6
0.9
2.6
6.1
8.7
3.6
2.6
2-Decenal
4-Hydroxy-2-Hexenal
2-Nonenal
2-Hexenal
GS-ONE
Crotonaldehyde
All-trans-Retinal
Farnesal
Results
Cloning, expression and purification of R1B10
13
49
The cDNA for R1B10 was amplified from the entire RNA samples
from the brain of female rats. The nucleotide sequence was con-
Acrolein

S. Endo et al. / Archives of Biochemistry and Biophysics 503 (2010) 230–237
233
Table 3
Kinetic constants for alcohols.
higher Km value than that for ONE, but did not exhibit significant
reductase activity towards ONOL.
Sugar and endogenous -dicarbonyl substrates of R1B10 are
listed in Table 2. The enzyme reduced - and -glyceraldehydes,
its 3-phosphate derivative and several tetroses more efficiently
than pentoses, although no reductase activity was observed for
kcat (minꢁ1
)
kcat/Km (minꢁ1 Mꢁ1
l )
a
Substrate
Km
(
lM)
D
L
Nerol
13
20
52
0.21
0.25
0.57
0.47
0.39
0.65
0.61
0.64
0.64
0.09
0.016
0.013
0.011
0.008
0.004
0.004
0.0004
0.0003
0.0003
0.0001
1-Decanol
1-Octanol
1-Nonanol
Geraniol
62
hexoses such as
D-glucose, D-galactose, D-fructose and D-glucuro-
100
160
1600
2400
2000
690
nate. -Dicarbonyls with alicyclic and aromatic rings (21-dehydro-
a
DHN
cortisol and isatin, respectively) were excellent substrates,
showing relatively high catalytic efficiency. The enzyme moder-
ately reduced AGE precursors such as 3-deoxyglucosone and meth-
ylglyoxal, while its reactivity for glyoxal was low. It should be
noted that the enzyme did not exhibit reductase activity towards
aromatic ketones (4-benzoylpyridine and 4-nitroacetophenone),
1-Hexanol
Benzyl alcohol
S-1-Indanol
Pyridine-3-methanol
direction, but were competitive inhibitors with respect to 1-octa-
nol in the reverse reaction (Ki values are 1.1 and 5.6 M, respec-
20-ketosteroids (progesterone and 3
ones) and PGs (D2 and E2).
a
-hydroxy-5
a
/b-pregnan-20-
l
tively). The inhibition patterns in the forward and reverse
directions are the same as those of human AKR1B10 by tolrestat
and quercetin [10,14]. In addition, the steady-state initial velocity
analysis of the 1-octanol oxidation by R1B10 indicated that the
reaction follows a mechanism with sequential binding of the coen-
zyme and substrate (data not shown), as the same kinetic mecha-
nism is reported for AKR1B10 [14]. Thus, the two inhibitors seem
to bind to the active site of the R1B10-coenzyme binary complex.
The reductase activity of R1B10 was activated by nonsteroidal
anti-inflammatory drugs (NSAIDs), which inhibit human AR and/
In the reverse reaction using NADP+ as the coenzyme, R1B10
oxidized several aromatic and aliphatic alcohols, including DHN
(Table 3). The Km value for NADP+ determined in the presence of
0.2 mM 1-octanol was 0.11 lM, which is lower than that for
NADPH. The kcat/Km values for the alcohol substrates were much
lower than those for the corresponding carbonyl products, when
the kinetic constants were compared in each oxidoreduction pair,
such as aliphatic alcohols vs aldehydes and benzyl alcohol vs benz-
aldehyde. No dehydrogenase activity was detected towards other
alicyclic alcohols (R-1-indanol, 2-cyclohexen-1-ol, benzene dihy-
or AKR1B10 [35]. Although sulindac (40 lM) and indomethacin
drodiol and naphthalene dihydrodiol), sugar alcohols (
and xylitol) and 20 -hydroxysteroids (20 -hydroxyprogesterone
and 20 -hydroxy-5 /b-pregnan-3-ones).
L
-threitol
(0.1 mM) showed weak inhibitions (<20%), other drugs (diclofenac,
ibuprofen, fenoprofen, flufenamic acid, mefenamic acid and meclo-
fenamic acid) enhanced the activity up to 1.3–2.0 folds at their
concentrations of 0.1 mM. Fig. 1A shows the dose-dependent acti-
vation by diclofenac and R-ibuprofen, whose isomer, S-ibuprofen
did not affect the enzyme activity. Diclofenac has a benzeneacetic
acid moiety, whose side-chain acetic acid is methylated in ibupro-
fen and fenoprofen (Fig. 1C). The structurally analogous cyclo-
hexylacetic acid, R-2-phenylpropionic acid and 3-phenylpropionic
acid also activated R1B10, whereas no significant activation was
observed by R-mandelic acid (2-hydroxy-2-phenylacetic acid), R-
3-phenyllactic acid, phenylpyruvic acid, phenoxyacetic acid and
hippuric acid (1 mM). In addition, R1B10 was activated by alde-
hyde reductase inhibitors (diphenic acid and valproic acid)
(Fig. 1B). As a benzoic acid structure is involved in molecules of
NSAIDs (flufenamic acid, mefenamic acid and meclofenamic acid)
and diphenic acid, benzoic acid and its analog cyclohexanoic acid
also activated the enzyme activity, but benzamide, benzyl alcohol,
cyclohexanol and salicylic acid did not show such stimulatory
effect. Valproate has a carboxylic group in the middle of the hydro-
carbon chain. The structurally analogous 2-ethylbutyric acid acti-
vated the enzyme at its high concentration range, but no
activation was observed by the addition of aliphatic carboxylic
a
a
a
a
Inhibitors and activators
The pyridine-3-aldehyde reductase activity of R1B10 was inhib-
ited by AR inhibitors, such as tolrestat, minalrestat, zopolrestat and
fidarestat (IC50 values were 7.0, 8.8, 14 and 19
flavonoids such as myricetin, daizein, quercitrin, and quercetin
(IC50 values were 8.2, 11, 35 and 46 M, respectively), and rat
AKR1B13 inhibitors such as benzbromarone, hexestrol and zearal-
enone (IC50 values of 5.0, 46 and 17 M, respectively). The IC50 val-
lM, respectively),
l
l
ues are higher than those reported with human AR, AKR1B10 [35]
and rat AKR1B13 [17]. Tolrestat and myricetin showed mixed-type
of inhibition with respect to pyridine-3-aldehyde in the reduction
Table 2
Substrate specificity for carbohydrates and
a-dicarbonyl compounds.
Substrate
Km
(lM)
kcat (minꢁ1
)
kcat/Km (minꢁ1 Mꢁ1
l )
Trioses
D
-Glyceraldehyde
96
220
390
3.0
2.8
1.7
0.031
0.013
0.004
L
-Glyceraldehyde
acids with saturated C4–C18 chains (10–500 lM), hydrophilic car-
boxylic acids (ascorbic acid, pyruvic acid and 3-hydroxybutyric
acid, 1 mM), bile acids (isolithocholic acid, lithocholic acid and
cholic acid, 10–100 lM), PGs (D2, E2 and F2 , 100 lM) and AR acti-
a
DL-Glyceraldehyde-3-P
Tetroses
-Erythrose
-Threose
D
150
210
350
314
3.3
2.2
2.9
1.8
0.022
0.010
0.008
0.006
D
vators (0.1 M LiSO4, 10 mM citrate and glucose-6-phosphate) [36].
The stimulatory effect by valproic acid was increased on elevat-
ing the pH, in contrast to that of the reductase activity by R1B10
(Fig. 1D). This, together with the pKa of valproic acid (4.3), suggests
the importance of its negatively charged carboxyl group for the
activation. The addition of valproic acid resulted in increases in
both Km for pyridine-3-aldehyde and kcat values, without apparent
change in the Km for NADPH. The lines of double reciprocal plots of
the activity versus pyridine-3-aldehyde concentration at fixed con-
centrations of valproic acid intersected below 1/[substrate] axis
(Fig. 1E). The replots of the reciprocal of change in the slope or
intercept of the respective primary reciprocal plot data versus
L
L
-Threose
-Erythrulose
Pentoses
-Xylulose
-Xylulose
-Arabinose
-Xylose
D
2000
3500
5900
1.2
1.5
0.78
0.84
0.0006
0.0004
0.0001
0.00007
L
L
D
12,000
a
-Dicarbonyls
21-Dehydrocortisol
Isatin
3.6
3.7
17
67
120
3.3
2.8
3.9
3.4
3.9
1.2
0.92
0.76
0.23
0.051
0.033
0.0005
3-Deoxyglucosone
Methylglyoxal
Diacetyl
Glyoxal
2300
1/[activator] were linear. The results are consistent with
a

234
S. Endo et al. / Archives of Biochemistry and Biophysics 503 (2010) 230–237
Fig. 1. Activation of the pyridine-3-aldehyde reductase activity of R1B10 by hydrophobic carboxylic acids. (A and B) Dose-dependent activation by cyclohexylacetic acid,
phenylpropionic acid and related compounds (a–e), and by valproic acid, benzoic acids and related compounds (f–j). (C) Structures of the activators (a–j) indicated in A and B.
(D) pH dependency of the activation by valproic acid. The pyridine-3-aldehyde reductase activities were determined in the absence (s) and presence of 0.5 mM valproic acid
(d), and the activation is shown as relative activity (N) to that without the activator. (E) Lineweaver–Burk analysis of the effect of valproic acid (VAL) on the reductase activity.
The activity was assayed with different concentrations of pyridine-3-aldehyde (P3A) in a fixed concentration of NADPH (0.1 mM). VAL concentrations: 0 (s), 0.125 (d), 0.25
(D) and 0.50 mM (N). Velocity is expressed in mU/mL. Replots of the 1/change in slope and intercept against 1/[VAL] are in the inset.
non-essential activation system, which implies the binding of one
activator molecule to a site of the enzyme [30]. The kinetic values
and AKR1B14) by RT-PCR using the primer specific to the cDNAs
for the four enzymes. As illustrated in Fig. 2A, the mRNA for
R1B10 was detected in many female rat tissues, of which it was
highly expressed in the brain and heart. A similar expression pat-
tern of the transcript in male rat tissues including the testis was
observed, and there was no sex difference in expression levels of
the mRNA (data not shown). The high expression in the brain
and heart was in contrast to tissue-specific AKR1B14 and ubiqui-
tous expression of the mRNAs for AR and AKR1B13, of which
AKR1B13 mRNA was not detected in the brain. The 37-kDa immu-
noreactive protein was observed in the heart and brain on Western
blotting using the anti-AKR1B10 polyclonal antibodies (Fig. 2B),
which cross-react with recombinant R1B10, but not with rat AR
and AKR1B14 (data not shown). RT-PCR analysis for the R1B10
mRNA in primary-cultured cells of rat coronary aorta showed that
of KA,
a and b for the non-essential activator valproic acid were
0.3 mM, 1.2 and 2.3, respectively. Valproic acid similarly activated
the rate of the reverse NADP+-linked 1-octanol oxidation, but af-
fected the Km values for both the substrate and coenzyme. The kcat
Km for 1-octanol, Km for NADP+ determined in the presence of
0.5 mM valproic acid were 0.84 minꢁ1, 240 and 0.6
M, respec-
,
l
tively, which are 1.6, 4.6 and 6.0 folds, respectively, of the kinetic
constants of the non-activated enzyme.
Tissue distribution
The tissue distribution of R1B10 was assessed and compared
with those of the AR (AKR1B4) and AR-like proteins (AKR1B13

S. Endo et al. / Archives of Biochemistry and Biophysics 503 (2010) 230–237
235
Fig. 2. Expression of R1B10 and its mRNA in male rat tissues and cells. (A) RT-PCR
analysis of expression of mRNAs for R1B10, AKR1B14, AKR1B13, rat AR and b-actin,
whose cDNA sizes amplified are 951, 445, 951, 951 and 764 base pairs, respectively.
Tissues: Br, brain; Lu, lung; He, heart; St, stomach; Li, liver; Ki, kidney; Ad, adrenal
gland; Si, small intestine; Co, colon; and Ov, ovary. (B) Western blot analysis using
the anti-human AKR1B10 antibodies. The cytosolic fractions (50
the homogenates of rat brain (lane 2) and heart (lane 3) were analyzed using the
recombinant R1B10 (0.1 g, in lane 1) as the positive control. (C) RT-PCR analysis of
lg proteins) from
l
expression of mRNA for R1B10 in rat primary-cultured endothelial cells (En) and
fibroblasts from the aorta (aF) and skin (sF), and rat glioma C6 cells (C6).
it was expressed in the fibroblasts but not in the endothelial cells
(Fig. 2C). The mRNA was also expressed in primary-cultured rat
skin fibroblasts and rat glioma C6 cell lines.
Protective effects against ONE and HNE in cells overexpressing R1B10
Fig. 3. Protective effects of R1B10 against ONE- and HNE-induced cytotoxicity
when overexpressed in BAECs. The cells were transfected for 48 h with the
expression vector alone (s) or the vector carrying R1B10 cDNA (d), and then
treated for 24 h with various concentrations of ONE or HNE. The dose-dependent
effects of ONE and HNE on viability of the R1B10-overexpressing cells are shown in
(A and B), respectively. The overexpression of R1B10 was verified by Western
blotting analysis (inset), where the samples 1, 2 and 3 are the control cell extract,
ONE, the best substrate for R1B10, is highly cytotoxic towards
cultured cells [37]. In order to evaluate the protective effect of
R1B10 against the cytotoxicity of ONE, we prepared the R1B10-
overexpressed BAECs, and compared the effect of ONE exposure
on the viability between the cells and control cells (transfected
with the empty vector). The high expression of cytosolic R1B10
in the cells transfected with the vector harboring the R1B10 cDNA
was confirmed by Western blotting using the antibody against hu-
man AKR1B10, in contrast to no immunopositive band in the ex-
tract of the control cells (Fig. 3A). The control cells were killed by
ONE in a dose-dependent manner. The toxicity against the cells
was attenuated by the overexpression of R1B10 at ONE concentra-
overexpressing cell extract (each 20
respectively.
lg) and recombinant R1B10 (0.1 lg),
respectively, based on high similarity in their sequences and
enzymatic properties [17–21]. Human AKR1B10 is distinct from
the rodent AR-like proteins, especially in its great ability to reduce
all-trans-retinal [10] and additional dehydrogenase activity to-
tions of less than 20 lM. We also examined the protective effect of
wards aromatic dihydrodiols [38] and 20a-hydroxysteroids [14].
the overexpression of the enzyme on the cytotoxicity of HNE
(Fig. 3B). As reported in other cells [37], HNE was less toxic to-
wards the control BAECs than ONE. While the enzyme reduced
HNE at less catalytic efficiency compared to ONE, significant pro-
tective effect against HNE-induced toxicity in the overexpressed
cells was observed.
The broad substrate specificity of R1B10 is similar to those of
the other rodent AR-like proteins, but clearly differs in the follow-
ing terms. (1) R1B10 exhibited reductase activity towards all-trans-
retinal, which is not reduced by rat AKR1B13 [17] and AKR1B14
(unpublished data). In this respect R1B10 resembles human
AKR1B10, but its catalytic efficiency for all-trans-retinal is much
lower than that of AKR1B10. In addition, R1B10 did not exhibit
Discussion
dehydrogenase activity towards aromatic dihydrodiols and 20a-
hydroxysteroids, a characteristic of human AKR1B10 [14,38]. (2)
When the kinetic constants for the substrates common to AKR1B7,
AKR1B8 and AKR1B13 [16,17,19,20] are compared, R1B10
exhibited lower Km values for most of the aliphatic aldehydes
and 3-deoxyglucosone than the three rodent AR-like proteins. (3)
R1B10 reduced short-chain (C3 and C4) aldoses with Km values
in the submillimolar range. Although glyceraldehyde is reduced
by the rodent AR-like proteins [16,17,20] and AR [7–9], the speci-
ficity for tetroses has not been reported with the mammalian
members of the AKR1B subfamily, except that rat AR reduces
In this study, we identified R1B10 encoded in the rat gene
Akr1b10 as a functional AKR that reduces a variety of aldehydes
and
a-dicarbonyl compounds, using NADPH as the preferable
coenzyme. R1B10 shows more than 83% overall amino acid
sequence identity not only with human AKR1B10, but also with
AKR1B7, AKR1B8, AKR1B13 and AKR1B14 (sequence identity of
85%, 84%, 84% and 88%, respectively). Although the detailed prop-
erties of AKR1B14 have not been reported [21], AKR1B13 and
AKR1B14 seem to be rat orthologs of mouse AKR1B8 and AKR1B7,

236
S. Endo et al. / Archives of Biochemistry and Biophysics 503 (2010) 230–237
L
-threose with a higher Km value (0.75 mM) than R1B10 [39]. (4)
tetroses generate superoxide anions in glycation reactions [55]
and -threose, one of the oxidation products of ascorbic acid, is also
suggested to be a potent glycation agent [56].
R1B10 showed less sensitivity to the AR inhibitors compared to
AKR1B8, AKR1B13 and human AKR1B10 [16,17,35], and is distinct
from AKR1B7 which is resistant to the AR inhibitors [20]. (5) R1B10
was activated by aliphatic, alicyclic and aromatic carboxylic acids.
Such activation by the carboxylic acids has not been reported with
other AR-like proteins.
Among the substrates of R1B10, ONE was the best substrate,
showing the highest kcat/Km value. ONE, a recently identified major
product of peroxidation of nꢁ6 polyunsaturated fatty acids, is
more reactive than another lipid peroxidation product, HNE
[6,37,40,41]. ONE is converted to GS-ONE (by nonenzymatic reac-
tion and glutathione-S-transferase [6,42]), the corresponding acid
(by aldehyde dehydrogenase [43]), ONOL (by human AR and
AKR1B10 [6,44]) and HNE (by human carbonyl reductase 1 [45]).
GS-ONE is efficiently reduced to GS-HNE by human AR [6]. The
Km value for ONE of R1B10 is much lower than those reported with
L
R1B10 was activated by hydrophobic carboxylic acids, and the
kinetic analysis of the activation by valproic acid indicates that this
compound acts as a non-essential activator, which binds to a site
distinct from the substrate and coenzyme binding sites. The results
of the comparative study on the efficacy of hydrophobic carboxylic
acids and related compounds provide the following specific struc-
tural requisites of the activator. (1) The existence of a negatively
charged carboxyl group in the activator molecule is necessary to
interact with the activator site of R1B10, because the aromatic
and alicyclic compounds without the free carboxyl group did not
activate. The pH dependency of the stimulatory effect indicates
that the ionized carboxyl group of the activator interacts with
the positively charged residue(s) in the activator-binding site. (2)
In addition to the carboxyl group, a hydrophobic moiety of the acti-
vator is required for its binding to the enzyme. In the aliphatic acti-
vators, the carboxyl group must be attached in the middle of the
more than 4-carbon hydrocarbon chain, because the activation
efficiency was decreased by shorting the hydrocarbon chain (as
shown in the cases of 2-ethylbutyric acid versus valproic acid)
and no stimulatory effect by fatty acids and PGs was observed. This
structural moiety is a constituent of benzoic acid, cyclohexanoic
acid and diphenic acid. In the other activators, the presence of
human AR (42
lM), AKR1B10 (300 lM) and carbonyl reductase
(346 M), so that R1B10 exhibits the highest kcat/Km value among
l
the reductases. Thus, R1B10 can be involved in the reduction of
ONE into less reactive ONOL at low intracellular concentration of
this aldehyde. This is supported by the protective effect against
the ONE-mediated cytotoxicity in the R1B10-overexpressing cells.
This protective effect was lower than that against HNE, despite of
the opposite catalytic efficiency of R1B10 for the two aldehydes.
The difference might be due to high reactivity of ONE over HNE
to thiols and cellular nucleophiles, electrophilicity of the reduced
product ONOL [6,46] and/or difference in signaling pathways lead-
ing to cell death between ONE and HNE [47].
the aromatic or alicyclic ring on the a- or b-carbon may be impor-
tant for the activation, as shown in the cases of diclofenac, cyclo-
hexylacetic acid, and 2- and 3-phenylpropionic acids. It should
be noted that the substituted group on the
a-carbon significantly
R1B10 also showed low Km values for other aldehydes such as
HNE, 4-hydroxy-2-hexenal, 1-hexanal, 2-nonenal and acrolein,
which are generated in lipid peroxidation under conditions of
oxidative stress [48]. Particularly, the Km for HNE is lower than
affects the activation. The presence of the methyl group did not af-
fect the activation, whereas the presence of the oxo and hydroxyl
group at this position attenuated the stimulatory effect. (3) In addi-
tion to the above structural requisites, nonpolar substituent on the
aromatic ring in the activator molecule probably contributes to the
affinity towards the activator site of the enzyme, as shown in the
difference in concentrations required for the activation among R-
ibuprofen, diclofenac and R-2-phenylpropionic acid. Thus, the acti-
vator-binding site on the enzyme may be composed of several
hydrophobic amino acids, in addition to the positively charged res-
idue(s) which interacts with the negatively charged carboxyl group
of the activator. Although we could not identify endogenous acti-
vators and the physiological significance of the activation is ob-
scure, the present study provides, for the first time, a novel
activation by hydrophobic carboxylic acids in the AKR1B subfamily
members.
those (9–300 lM) of ARs and other rodent AR-like proteins
[5,9,16,17,20,44], and comparable to that of the most efficient
HNE reductase, rat AKR1C15 [33]. The contribution of R1B10 to
the detoxification of HNE is evidenced by the significant protective
effect against the HNE-mediated cytotoxicity in the R1B10-over-
expressing cells. Unlike ONE and HNE produced from peroxidation
of n ꢁ 6 polyunsaturated fatty acids, 4-hydroxy-2-hexenal is a ma-
jor reactive aldehyde derived from peroxidation of n ꢁ 3 polyun-
saturated fatty acids that are rich in brain and retina [49]. The
Km value for 4-hydroxy-2-hexenal of R1B10 is much lower than
those of aldehyde dehydrogenases (Km = 28–50
known reductases, bovine AR (Km = 40 M) [49] and AKR1B8
(Km = 155 M) [16]. Furthermore, R1B10 exhibited a low Km value
lM) and two
l
l
for crotonaldehyde, which is detected as protein adducts in human
brain with Alzheimer’s disease [50]. The high reactivity towards
the reactive lipid-derived aldehydes suggests that R1B10 is impli-
cated in detoxification of the reactive aldehydes derived from lipid
peroxidation, especially in rat brain and heart.
Acknowledgments
This work was supported by a Grant-in-Aid for Young Scientists
from the Japan Society for the Promotion of Science.
R1B10 reduced 3-deoxyglucosone, glyoxal and methylglyoxal,
which are generated in the course of nonenzymatic glycation and
sugar metabolism, and result in the formation of AGEs [51,52].
The endogenous compounds and major AGE precursors in food
(furfural and hydroxyfurfural) [53] are reduced by mouse, rat and
human members in the AKR superfamily [17,54]. When the kinetic
constants for the AGE precursors are compared among R1B10 and
these members of the superfamily, R1B10 shows low Km values for
all the precursors. In particular, the Km value for 3-deoxyglucosone
is the lowest, and its kcat/Km value is comparable to those of ARs
and aldehyde reductases, which are reported to be efficient
3-deoxyglucosone reductases [8]. Thus, R1B10 may play a role in
preventing AGE formation by reducing the AGE precursors. In addi-
tion, the ability to reduce short-chain aldoses found in the present
study may also prevent the AGE formation, because trioses and
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