Characterization of rabbit aldose reductase-like protein with 3β-hydroxysteroid dehydrogenase activity

Article abstract of DOI:10.1016/j.abb.2012.07.012

In this study, we isolated the cDNA for a rabbit aldose reductase-like protein that shared an 86% sequence identity to human aldo-keto reductase (AKR)¹ 1B10 and has been assigned as AKR1B19 in the AKR superfamily. The purified recombinant AKR1B19 was similar to AKR1B10 and rabbit aldose reductase (AKR1B2) in the substrate specificity for various aldehydes and α-dicarbonyl compounds. In contrast to AKR1B10 and AKR1B2, AKR1B19 efficiently reduced 3-keto-5α/β-dihydro-C19/C21/C24-steroids into the corresponding 3β-hydroxysteroids, showing K_m of 1.3-9.1 μM and k_cat of 1.1-7.6 min^-1. The stereospecific reduction was also observed in the metabolism of 5α- and 5β- dihydrotestosterones in AKR1B19-overexpressing cells. The mRNA for AKR1B19 was ubiquitously expressed in rabbit tissues, and the enzyme was co-purified with 3β-hydroxysteroid dehydrogenase activity from the lung. Thus, AKR1B19 may function as a 3-ketoreductase, as well as a defense system against cytotoxic carbonyl compounds in rabbit tissues. The molecular determinants for the unique 3-ketoreductase activity were investigated by replacement of Phe303 and Met304 in AKR1B19 with Gln and Ser, respectively, in AKR1B10. Single and double mutations (F303Q, M304S and F303Q/M304S) significantly impaired this activity, suggesting the two residues play critical roles in recognition of the steroidal substrate.

Full text of DOI:10.1016/j.abb.2012.07.012

Archives of Biochemistry and Biophysics 527 (2012) 23–30
Contents lists available at SciVerse ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
Characterization of rabbit aldose reductase-like protein with 3b-hydroxysteroid
dehydrogenase activity
a
a
a
b
c
Satoshi Endo a, , Toshiyuki Matsunaga , Sho Kumada , Airi Fujimoto , Satoshi Ohno , Ossama El-Kabbani ,
⇑
Dawei Hu d, Naoki Toyooka d, Jun’ichi Mano e, Kazuo Tajima f, Akira Hara a
a Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan
b Faculty of Engineering, Gifu University, Gifu 501-1193, Japan
c Monash Institute of Pharmaceutical Sciences, Monash University, Victoria 3052, Australia
d Graduate School of Science and Technology for Research, Graduate School of Innovative Life Science, University of Toyama, Toyama 930-8555, Japan
e Science Research Center, Yamaguchi University, Yamaguchi 753-8515, Japan
f Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we isolated the cDNA for a rabbit aldose reductase-like protein that shared an 86% sequence
identity to human aldo–keto reductase (AKR)1 1B10 and has been assigned as AKR1B19 in the AKR super-
family. The purified recombinant AKR1B19 was similar to AKR1B10 and rabbit aldose reductase (AKR1B2) in
Received 15 June 2012
and in revised form 9 July 2012
Available online 1 August 2012
the substrate specificity for various aldehydes and
AKR1B2, AKR1B19 efficiently reduced 3-keto-5 /b-dihydro-C19/C21/C24-steroids into the corresponding
3b-hydroxysteroids, showing Km of 1.3–9.1
M and kcat of 1.1–7.6 minꢀ1. The stereospecific reduction
was also observed in the metabolism of 5 - and 5b-dihydrotestosterones in AKR1B19-overexpressing cells.
a-dicarbonyl compounds. In contrast to AKR1B10 and
a
Keywords:
AKR1B10
Aldo–keto reductase
Aldose reductase
3b-Hydroxysteroid dehydrogenase
4-Oxo-2-nonenal
l
a
The mRNA for AKR1B19 was ubiquitously expressed in rabbit tissues, and the enzyme was co-purified with
3b-hydroxysteroid dehydrogenase activity from the lung. Thus, AKR1B19 may function as a 3-ketoreduc-
tase, as well as a defense system against cytotoxic carbonyl compounds in rabbit tissues. The molecular
determinants for the unique 3-ketoreductase activity were investigated by replacement of Phe303 and
Met304 in AKR1B19 with Gln and Ser, respectively, in AKR1B10. Single and double mutations (F303Q,
M304S and F303Q/M304S) significantly impaired this activity, suggesting the two residues play critical roles
in recognition of the steroidal substrate.
Ó 2012 Elsevier Inc. All rights reserved.
Introduction
3b-HSD/D5
VII, are identified, and oxidize 3b-hydroxy-
and 3b-hydroxy-
5-C27-steroids. In addition, several human
NADPH-dependent enzymes, belonging to the aldo–keto reduc-
tase (AKR) superfamily (http://www.med.upenn.edu/akr/) [2]
and the short-chain dehydrogenase/reductase (SDR) superfamily
–
D4 isomerase isoenzymes, type-I, type-II and type-
D
5-C19/C21-steroids
3b-Hydroxysteroid dehydrogenase (HSD1)/D5
–
D4 isomerase is
D
a NAD+-dependent membrane-bound enzyme that is responsible
for the oxidation and isomerization of
D
5-3b-hydroxysteroid pre-
cursors into
D
4-3-ketosteroids (Fig. 1), thus catalyzing an essen-
tial and irreversible step in the biosynthesis of all classes of
active steroid hormones and bile acids [1]. In humans, three
[3], have been reported to reduce 3-keto-5
into the corresponding 3b-hydroxysteroids (Fig. 1). Cytosolic
20 -HSD (AKR1C1), 3 -HSD type-3 (AKR1C2), 17b-HSD type-5
(AKR1C3), and 3 -HSD type-1 (AKR1C4) non-stereospecifically re-
duce 5 -dihydrotestosterone into its 3 - and 3b-ol forms, and of
the four enzymes AKR1C1 exhibits the most efficient 3b-HSD
activity [4]. The stereospecific reduction of 3-keto-C19/C21
steroids into 3b-hydroxysteroids is catalyzed by membrane-
associated 17b-HSD type-7 [5] and peroxisomal dehydrogenase/
reductase (SDR family) member 4 (DHRS4) [6] that belong to
the SDR superfamily. These NADPH-dependent enzymes are
thought to be involved in the synthesis of the following
a/b-dihydrosteroids
a
a
a
⇑
Corresponding author. Fax: +81 58 230 8105.
E-mail address: sendo@gifu-pu.ac.jp (S. Endo).
Abbreviations used: HSD, hydroxysteroid dehydrogenase; AKR, aldo–keto reduc-
a
a
1
-
tase; SDR, short-chain dehydrogenase/reductase; DHRS4, dehydrogenase/reductase
(SDR family) member 4; AR, aldose reductase; SI, sequence identity; ONE, 4-oxo-2-
nonenal; AKR1B19, rabbit aldose reductase-like protein; PHPC, (Z)-2-(phenylimino)-
7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide; HAHE, 3-(4-hydroxy-2-
methoxyphenyl)acrylic acid 3-(3-hydroxyphenyl)propyl ester; TBE, 6-tert-butyl-2,3-
epoxy-5-cyclohexene-1,4-dione; GS-ONE, glutathione adduct of ONE; RT, reverse
transcription; bp, base pair; U, unit; LC/MS, liquid chromatography/mass spectrom-
etry; BAEC, bovine aortic endothelial cell; FBS, fetal bovine serum.
biologically active 3b-hydroxy-5a/b-dihydro-C19/C21-steroids.
0003-9861/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.abb.2012.07.012

24
S. Endo et al. / Archives of Biochemistry and Biophysics 527 (2012) 23–30
Materials and methods
Materials
Steroids were obtained from Steraloids (Newport, RI); 4-oxo-2-
nonenal (ONE), 4-hydroxy-2-nonenal, 4-hydroxy-2-hexenal and
prostaglandins were from Cayman Chemical (Ann Arbor, MI); res-
ins for column chromatography were from Amersham Biosciences
(Piscataway, NJ); and (Z)-2-(4-methoxyphenylimino)-7-hydroxy-
N-(pyridin-2-yl)-2H-chromene-3-carboxamide (PHPC) was from
Asinex (Moscow, Russia). Pfu DNA polymerase was purchased from
Stratagene; a pCold I expression vector and Taq DNA polymerase
were from Takara (Kusatsu, Japan); and pCR2.1 plasmid, restriction
enzymes, RACE kits, Lipofectamine 2000 reagent and Escherichia
coli BL21 (DE3) pLysS were from Invitrogen (Carlsbad, CA). AR
inhibitors were kindly donated by Dr. P.F. Kador (University of
Nebraska Medical Center).
Fig. 1. Conversion catalyzed by NAD+-dependent 3b-HSD/D5 4 isomerase (A) and
–D
NADPH-dependent enzymes with reductive 3b-HSD activity (B).
Synthesis of inhibitor and substrates
3-(4-Hydroxy-2-methoxyphenyl)acrylic acid 3-(3-hydroxy-
phenyl)propyl ester (HAHE, a human AKR1B10 inhibitor) [23], 6-
tert-butyl-2,3-epoxy-5-cyclohexene-1,4-dione (TBE) [24], isocap-
roaldehyde [25] and geranylgeranial [26] were synthesized as de-
scribed previously. 4-Oxo-2-nonenol, 4-hydroxy-2-nonenol and a
glutathione adduct of ONE (GS-ONE) were prepared by the meth-
ods of Doorn et al. [27]. 4-Oxo-2-hexenal was synthesized accord-
ing to the method of Grée et al [28]. Briefly, to a stirred solution of
2-ethylfuran (1 g) and N-bromosuccinimide (2.72 g) in tetrahydro-
furan–acetone–water (10:8:2; v/v, 20 mL) at ꢀ20 °C was added
dropwise 1.62 g of pyridine and kept stirred for 3 h. The tempera-
ture was then allowed to 25 °C, and the mixture was stirred over-
night. A 0.5 M HCl aqueous solution (20 mL) was then added. The
product was extracted into ether (20 mL) three times. The solvent
was dried over anhydrous sodium sulfate, and evaporated under
reduced pressure. The residue was chromatographed on a silica
gel column with pentane–ether (85:15; v/v) as the eluent to yield
pure 4-oxo-2-hexenal (300 mg). Identity and purity of 4-oxo-2-
hexenal were confirmed with NMR [29].
5a-Androstane-3b,17b-diol strongly binds to the estrogen recep-
tor b, regulates brain function, and modulates the growth of
prostate and its cancer [7–10]. 3b-Hydroxypregnanes are preg-
nenolone sulfate-like antagonists of
c-aminobutyric acid type-A
receptor [11], and 5b-pregnan-3b-ol-20-one shows stimulatory
action on preoptic neurons [12].
In rats and mice, in addition to the NAD+-dependent 3b-HSD/
D5 D4 isomerase, its NADPH-dependent isoenzymes (type-III,
–
type-IV and type-V) are expressed and function as 3-ketosteroid
reductases which produce 3b-hydroxy-C19/C21-steroids [1]. Such
a NADPH-dependent isoenzyme has not been identified in human
tissues [1,13], where human AKRs (1C1–1C4), 17b-HSD type-7 and
DHRS4 exhibit the reductive 3b-HSD activity as described above. In
contrast to the human 17b-HSD type-7 and DHRS4, mouse 17b-
HSD type-7 does not reduce 3-keto-C19-steroids [5], and rat and
rabbit DHRS4s exhibit 3a-HSD activity [14]. Rabbit 20a-HSD
(AKR1C5), which corresponds to human AKR1C1, exhibits 3
a
-
HSD activity towards 3-ketosteroids [15]. Thus, there is a clear spe-
cies difference in function of enzymes that act as reductive 3b-
HSDs.
CDNA isolation and site-directed mutagenesis
Recently, mouse aldose reductase (AR)-like protein (AKR1B7)
has been reported to reduce 3-keto bile acids into the correspond-
ing 3b-hydroxy bile acids, in addition to their inherent roles in the
metabolism of aldehydes [16]. In contrast, 3-ketosteroids including
bile acids are not reduced by human AR (AKR1B1) and AR-like pro-
tein (AKR1B10) [17] and rat AR-like proteins (AKR1B13 [18],
AKR1B14 [19] and AKR1B18 [20]), and rather inhibit AKR1B1 and
AKR1B10 [17]. The human and rodent AR-like proteins also differ
in their kinetic constants for common carbonyl substrates [16–
20] and reactivity to prostaglandins [21,22]. Current rabbit geno-
mic analysis has predicted only one gene encoding an AR-like pro-
tein that shares more than 80% amino acid sequence identity (SI)
with the human and rodent AR-like proteins. In this paper, we iso-
lated the cDNA for the rabbit AR-like protein that has been as-
signed as AKR1B19 in the AKR superfamily, and examined the
enzymatic properties of the recombinant AKR1B19 and tissue dis-
tribution. The data indicated that AKR1B19 acts not only as a
reductase for reactive carbonyl compounds derived from lipid
peroxidation like AR-like proteins of other species, but also as a
The cDNAs for AKR1B19 and rabbit AR (AKR1B2) [30] were iso-
lated from the total RNA preparation of lungs of male Japanese
white rabbits by reverse transcription (RT)-PCR. The preparation
of total RNA, RT, and DNA techniques followed the standard proce-
dures described by Sambrook et al. [31]. PCR was performed with
Pfu DNA polymerase and a pair of sense and antisense primers,
which contain NdeI and EcoRI sites. The primer sequences and
PCR conditions are summarized in Table S1 (Supplementary Data).
The PCR products were purified, digested with the two restriction
enzymes, and ligated into the pCold I vectors that had been di-
gested with the two restriction enzymes. The insert of the cloned
cDNA was sequenced by using a Beckman CEQ8000XL DNA se-
quencer, and was confirmed to encode the 316-amino acid se-
quences of AKR1B19 and AKR1B2 fused to the N-terminal 6-His
tag. The 30- and 50-untranslated regions of the AKR1B19 mRNA
were generated by using 30- and 50-RACE kits and the gene-specific
primers. The fragments were subcloned into the pCR2.1 plasmids
and sequenced as described above. The 1324-base pair (bp) se-
quence of the cDNA including the poly(A) sequence was deposited
in DDBJ database with the accession number AB724112.
superior reductive 3b-HSD for 3-keto-5a/b-dihydro-C19/C21/C24-
steroids. Therefore, the molecular determinant for 3b-HSD activity
was also investigated by site-directed mutagenesis of residues of
this enzyme.
Mutagenesis was performed using a QuickChange site-directed
mutagenesis kit (Stratagene) and the pCold I expression plasmid
harboring the cDNA for AKR1B19 as the template according to

S. Endo et al. / Archives of Biochemistry and Biophysics 527 (2012) 23–30
25
the protocol described by the manufacturer. The primer pair used
for the mutagenesis was composed of sense and antisense oligonu-
cleotides to give L116F, F303Q, M304S and F303Q/M304S mutant
AKR1B19s (Table S1, Supplementary Data). The coding regions of
the cDNAs in the expression plasmids were sequenced in order
to confirm the presence of the desired mutation and ensure that
no other mutation had occurred.
equation. The kinetic constants for NADPH and NADP+ were deter-
mined in the presence of 0.2 mM pyridine-3-aldehyde and 40
l
M
5b-androstan-3b-ol-17-one, respectively, as the substrates. After
the IC50 (inhibitor concentrations required for 50% inhibition) val-
ues were determined with pyridine-3-aldehyde as the substrate,
the kinetic studies in the presence of three concentrations of an
inhibitor (0.2–1 ꢁ IC50) were carried out in a similar manner in
both pyridine-3-aldehyde reduction and 5b-androstan-3b-ol-17-
one oxidation. The inhibitor constants, Kis and Kii, were calculated
from the replots of the slopes and intercepts, respectively, of the
double reciprocal plot versus the inhibitor concentration. The ki-
netic constants and IC50 are expressed as the means of two deter-
minations or the means SD of three determinations.
Purification of enzymes
The recombinant AKR1B19, its mutant enzymes, AKR1B2 and
human AKR1B10 were expressed in E. coli BL21 (DE3) pLysS cells
transformed with the expression plasmids harboring their cDNAs
as described previously [17]. The enzymes were purified from
the cell extracts using a nickel-charged Sepharose 6FF resin accord-
ing to the manufacturer’s manual. The enzyme fraction was con-
centrated by ultrafiltration and dialyzed against 10 mM Tris–HCl
buffer, pH 8.0, containing 1 mM dithiothreitol, 0.5 mM EDTA and
20% (v/v) glycerol. Purity was confirmed by SDS–PAGE, and protein
concentration was determined by Bradford’s method using bovine
serum albumin as the standard [31]. The identity of the purified
recombinant protein with AKR1B19 encoded in the cDNA was
verified by the mass spectrometry analysis of its 15 lysylendopep-
tidase-digested peptides using a Bruker–Franzen Mass Spectrome-
try System Ultraflex TOF/TOF [32].
AKR1B19 was also partially purified at 4 °C from rabbit lung.
The rabbit lung (4 g) was homogenized with four volumes of
50 mM Tris–HCl, pH 7.4, containing 5 mM EDTA and 0.15 M KCl.
The homogenate was centrifuged at 105,000g for 1 h, and the
supernatant fraction was subjected to (NH4)2SO4 fractionation
(35–75% saturation). The protein fraction was dissolved in
10 mM Tris–HCl, pH 8.0, containing 5 mM EDTA (Buffer A) plus
0.15 M KCl, and then applied to a Sephadex G-100 column
(3 ꢁ 70 cm) equilibrated with the buffer. The fraction with both
pyridine-3-aldehyde reductase and 5b-androstan-3b-ol-17-one
dehydrogenase activities was applied to a Q-Sepharose column
(2 ꢁ 15 cm) that had been equilibrated with Buffer A. After the col-
umn was washed with the buffer, the adsorbed proteins were
eluted with a linear gradient of 0–0.1 M NaCl in the buffer.
Product identification
To identify reaction products, reduction was conducted in a 2.0-
mL system containing 0.2 mM NADPH, substrate (0.05–1 mM), en-
zyme (1–20 lg) and 0.1 M potassium phosphate, pH 7.4. The sub-
strate and products were extracted into 4 mL ethyl acetate 5–
20 min after the reaction was started at 37 °C. The steroidal prod-
ucts were identified by TLC and liquid chromatography/mass spec-
trometry (LC/MS) using a Chiralcel OJ-H 5 lm column [6]. The TLC
chromatogram was developed in chloroform–acetone (9:1, v/v)
twice, and the products and co-chromatographed authentic ste-
roids were visualized by spraying with ethanol–H2SO4 (1:1, v/v)
solution and heating at 110 °C for 1 h. The reduced products of
ONE were also analyzed by the TLC method [27].
Tissue distribution analysis
The total RNA samples were prepared from the tissues of male
rabbits [31]. The total RNA samples were subjected to RT-PCR
using Taq DNA polymerase and primers to amplify cDNAs for
AKR1B19, AKR1B2 and rabbit
D
4-3-ketosteroid 5b-reductase
(AKR1D3, accession no. AB023951). The cDNA for rabbit b-actin
was also amplified as an internal control with the specific primers.
The primer sequences and PCR conditions are described in Table S1
(Supplementary Data). The PCR products were separated by aga-
rose gel electrophoresis, and revealed with ethidium bromide.
Assay of enzyme activity
Cell culture experiments
Reductase and dehydrogenase activities of AKR1B19, AKR1B2
and AKR1B10 were assayed by measuring the rate of change in
NAD(P)H absorbance (at 340 nm) and its fluorescence (at 455 nm
with an excitation wavelength of 340 nm), respectively. The stan-
dard reaction mixture for the reductase activity consisted of
0.1 M potassium phosphate, pH 7.4, 0.1 mM NADPH, 0.2 mM pyri-
dine-3-aldehyde and enzyme, in a total volume of 2.0 mL. In the as-
say for the dehydrogenase activity, 0.25 mM NADP+ was used as
the coenzyme. The reductase and dehydrogenase activities of the
lung extract and fractions during the enzyme purification were as-
Bovine aortic endothelial cells (BAECs) were generous gift from
Taisho Pharmaceutical Co. (Saitama, Japan), and cultured in Dul-
becco’s modified Eagle medium supplemented with 10% fetal bo-
vine serum, penicillin (100 U/mL) and streptomycin (100 lg/mL)
at 37 °C in a humidified incubator containing 5% CO2. The con-
struction and transfection of the pGW1 plasmids harboring the
cDNA for AKR1B19 into BAECs were performed as described pre-
viously [34]. Briefly, the cDNA was amplified by polymerase chain
reaction (PCR) using Pfu DNA polymerase and a pair of the for-
ward and reverse primers (Table S1, Supplementary Data), which
contain underlined EcoRI and SalI sites, respectively. The PCR
products were ligated into the pGW1 vectors that had been
digested with the two restriction enzymes. The expression con-
structs were transiently transfected with the expression plasmids
using the Lipofectamine 2000 reagent. The transfected cells were
maintained in the medium containing 2% FBS for 24 h, and then
used for experiments. The cell extract was subjected to the assay
of the pyridine-3-aldehyde reductase activity. In the analysis of
cellular 3-ketosteroid metabolism, the cells were plated into a
sayed with 0.2 mM pyridine-3-aldehyde and 50 lM 5b-androstan-
3b-ol-17-one, respectively, as the substrates. The steroids and
other water-insoluble compounds were dissolved in methanol,
and added into the reaction mixture, in which the final concentra-
tion of methanol was less than 1.25%. The concentration of meth-
anol did not affect the activities of the enzyme. The reductase
activity for all-trans-retinal was measured by the method of Parés
and Julià [33], except that 0.1 M potassium phosphate, pH 7.4, con-
taining 0.01% Tween 80 was added as the buffer. One unit (U) of
enzyme activity was defined as the enzyme amount that catalyzes
the reduction of 1
l
mol of NADPH or retinal per min at 25 °C.
6-cm dish and cultured in the medium containing 50 lM 5a-
The apparent Km and kcat values were determined over a range
of five substrate concentrations at a saturating concentration of
coenzyme by fitting the initial velocities to the Michaelis–Menten
dihydrotestosterone or 5b-dihydrotestosterone for 6 h. The
steroid and its metabolites in the medium were extracted into
8 mL ethyl acetate, and the organic layer was taken and

26
S. Endo et al. / Archives of Biochemistry and Biophysics 527 (2012) 23–30
Table 1
evaporated. The 3-hydroxy metabolites of dihydrotestosterone
were analyzed by the above LC/MS method, in which the eluted
metabolites were monitored using the negative ion mode of m/
z 293.5.
Substrate specificity for carbonyl compounds of AKR1B19.
Substrate
Km
(l
M)
kcat (minꢀ1
)
kcat/Km
(minꢀ1 Mꢀ1
l )
Aldehydes
ONE
GS-ONE
trans-2-Nonenal
Geranylgeranial
Farnesal
1.0
5.8
8.0
1.1
1.1
28
15
9.0
52
4.0
30
28
22
30
4.6
2.8
2.0
Results
2.2
2.0
22
7.1
3.7
20
1.4
2.5
12
Cloning, expression and purification of AKR1B19
1.8
Acrolein
0.77
0.47
0.41
0.38
0.35
0.19
0.13
0.11
The full-length cDNA (1324-bp) for AKR1B19 was isolated by
RT-PCR from the total RNA sample of rabbit lung. The nucleotide se-
quence of the coding region was identical to that of the mRNA for
Oryctolagus cuniculus AKR1B10 (Accession No. XM_002711986)
predicted from the rabbit genomic analysis, with exception of three
nucleotide replacements (135T ? C, 264G ? T, 459A ? G) that do not
affect the amino acid sequence of AKR1B19. 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
AKR1B19 was purified to homogeneity by detecting the 37-kDa
protein on the SDS–PAGE analysis. The sequences (composed of a
total 238 residues) of 15 lysylendopeptidase-digested peptides de-
rived from the purified enzyme were identical to those deduced
from the cDNA for AKR1B19 (Fig. S1, Supplementary Data).
4-Hydroxy-2-nonenal
Isocaproaldehyde
Crotonaldehyde
all-trans-Retinal
4-Hydroxy-2-hexenal
trans-2-Hexenal
4-Oxo-2-hexenal
13
87
37
3.9
a
-Dicarbonyls
Isatin
8.6
3.3
11
18
0.38
0.15
0.080
0.066
3-Deoxyglucosone
Methylglyoxal
Diacetyl
74
222
455
30
Monosaccharides
1220
2100
1100
13
15
5.3
0.011
0.007
0.005
D
-Glyceraldehyde
-Glyceraldehyde
-Threose
L
D
Xenobiotics
Substrate specificity of AKR1B19
TBE
1.6
7.0
11
15
346
141
35
77
43
11
12
2.2
22
11
3.9
0.73
0.035
0.016
a
TBQ
The purified AKR1B19 exhibited NADPH-linked reductase activ-
ity (1.1 U/mg) towards 0.2 mM pyridine-3-aldehyde at pH 7.4,
which was higher than the specific activity (0.25 U/mg) of recom-
binant rabbit AR (AKR1B2) determined under the same conditions.
The optimum pH for the activity of AKR1B19 was observed be-
tween pH 6.2 and 6.4, while AKR1B2 showed a broad pH optimum
from pH 6.2 to 6.8. AKR1B19 showed high coenzyme preference to
Pyridine-3-aldehyde
Benzaldehyde
3-Nitroacetophenone
a-Tetralone
a
TBQ, 2-tert-butyl-1,4-benzoquinone.
Table 2
NADPH. The Km and kcat values for NADPH were 1.0
lM and
Substrate specificity for steroids.
44 minꢀ1, respectively, whereas the NADH (100
lM)-linked reduc-
Substrate
Km
M)
kcat
(minꢀ1
kcat/Km
tase activity was only 18% of the NADPH-linked activity. Therefore,
the substrate specificity of AKR1B19 was examined using the pref-
erable coenzyme NADPH at a physiological pH of 7.4, and com-
pared with those of AKR1B2.
(l
)
(minꢀ1 Mꢀ1
l )
Reduction
5b-Pregnane-21-ol-3,20-
dione
2.4
5.5
2.3
AKR1B19 reduced endogenous aliphatic aldehydes with various
carbon chain lengths from acrolein to geranylgeranial (Table 1).
Among them, ONE was the best substrate showing the highest cat-
alytic efficiency (kcat/Km value). In the product analysis of the
reduction of ONE by TLC, its alcohol, 4-oxo-2-nonenol, was de-
tected as only one product. Although no reductase activity for 4-
oxo-2-nonenol by the enzyme was detected in the spectrophoto-
metric activity assay, GS-ONE was reduced with a sixfold higher
Km value than that for ONE. The enzyme also reduced endogenous
Dehydrolithocholic acid
5b-Dihydrotestosterone
5b-Androstane-3,17-dione
5b-Pregnan-20b-ol-3-one
5b-Pregnane-3,20-dione
1.3
3.5
4.1
3.0
5.5
2.2
9.1
3.1
6.8
7.6
4.0
6.4
2.4
5.2
2.3
1.9
1.9
1.3
1.2
1.1
0.57
5
a
-Dihydrotestosterone
-Pregnane-21-ol-3,20-
dione
5a
5a-Pregnane-3,20-dione
8.8
7.3
6.4
7.6
4.9
5.0
2.7
2.2
2.6
1.1
0.57
0.37
0.35
0.34
0.22
5b-Dihydrocorticosterone
5b-Dihydrocortisone
5a-Androstane-3,17-dione
a
-dicarbonyl compounds, and xenobiotic aromatic aldehydes, p-
quinones (TBE and 2-tert-butyl-1,4-benzoquinone) and ketones
(3-nitroacetophenone and -tetralone), of which the two p-qui-
nones were excellent substrates, showing high kcat/Km values.
AKR1B19 showed low activity towards - and -glyceraldehydes
-threose, but no reductase activity was observed for -glucose
-xylose that were reduced by AKR1B2. The comparison of the
5b-Cholanic acid-3,7-dione
a
Oxidation
Isolithocholic acid
1.6
9.4
7.8
2.2
5.2
3.2
1.4
0.55
0.41
D
L
5b-Androstane-3b,17b-diol
5b-Pregnan-3b,21-diol-20-
one
5b-Androstan-3b-ol-17-one
5b-Pregnan-3b-ol-20-one
5b-Pregnane-3b,20b-diol
and
and
D
D
D
7.3
2.7
4.9
2.7
2.6
0.23
0.060
0.24
0.36
0.22
0.21
0.11
0.079
0.060
0.025
kinetic constants for the above substrates between AKR1B19 and
AKR1B2 is summarized in Table S2 (Supplementary Data). Only
4-hydroxy-2-hexenal, 4-oxo-2-hexenal, methylglyoxal and mono-
saccharides were efficiently reduced by AKR1B2, whereas the
kcat/Km values for other substrates of AKR1B19 were higher than
those of AKR1B2.
22
13
22
3.1
1.0
9.6
5b-Pregnane-3b,20
a-diol
5a-Cholanic acid-3b-ol
Geranylgeraniol
Farnesol
Since AKR1B19 reduced the xenobiotic ketones, its substrate
specificity for endogenous ketones, prostaglandins and ketoste-
1.3–9.1
activity was observed for 17- and 20-ketosteroids,
roids (testosterone, 4-androstene-3,17-dione and progesterone),
l
M and kcat values of 1.1–7.6 minꢀ1 (Table 2). No reductase
4-3-ketoste-
D
roids, was examined. The enzyme reduced 3-keto-5
a/b-dihydro-
C19/C21-steroids and 3-keto bile acids, showing Km values of

S. Endo et al. / Archives of Biochemistry and Biophysics 527 (2012) 23–30
27
5b-androstane-3b,17b-diol, 5b-androstan-3b-ol-17-one and 5b-
pregnan-3b-ol-20-one, respectively, by TLC. To further confirm
the stereo-specific reduction of 3-ketosteroids by AKR1B19, we
examined the metabolism of 5a-dihydrotestosterone and 5b-dihy-
drotestosterone in the BAECs transfected with the expression plas-
mid harboring the cDNA for the enzyme. The overexpression of
AKR1B19 was validated by detecting fivefold higher pyridine-3-
aldehyde reductase activity in the extract of the transfected cells
compared to the activity in the control cells transfected with the
vector alone. In the reduction of 5
a-dihydrotestosterone, only
one product, 5 -androstane-3b,17b-diol, was formed, as shown
a
in the LC/MS chromatogram (Fig. 2A). The incubation with 5b-
dihydrotestosterone in the transfected cells produced both 5b-
androstane-3b,17b-diol and 5b-androstane-3
jor and minor products, respectively, in contrast to the formation
of only 5b-androstane-3 ,17b-diol in the control cells due to their
intrinsic reductive 3 -HSD activity for 5b-dihydrotestosterone
a,17b-diol as the ma-
a
a
(Fig. 2B). The addition of the AKR1B19 inhibitor, HAHE (Table 3),
into the medium of the transfected cells significantly decreased
the amount of 5b-androstane-3b,17b-diol, but did not have any ef-
fect on that of 5b-androstane-3a,17b-diol, being indicative of the
stereo-selective reduction of the 3-ketosteroids by AKR1B19.
Inhibitor sensitivity
The reductase activity of AKR1B19 was inhibited by inhibitors
of human AKR1B10 [23,35–37] and AR (AKR1B1), and the inhibi-
tory effects were compared with those of rabbit AR, AKR1B2 (Ta-
ble 3). PHPC is a potent and nonselective inhibitor of the two
human enzymes [36], and most potently inhibited both AKR1B19
and AKR1B2. HAHE, a selective inhibitor of human AKR1B10 [23],
showed the highest selectivity ratio of AKR1B19 to AKR1B2 among
the inhibitors tested. Minalrestat was
a potent inhibitor of
Fig. 2. LC/MS analysis of reduced products of 5
AKR1B19-overexpressed BEACs. The cells were cultured with 50
6 h, and the products and remaining substrate in the medium were extracted and
subjected to LC/MS. (A) Reduction of 5 -dihydrotestosterone (5 -DHT). Chromato-
grams: (s) authentic 5 -androstane-3 ,17b-diol (3 ) and 5 -androstane-3b,17b-
a
- and 5b-dihydrotestosterones in
AKR1B19, but all AR inhibitors including minalrestat inhibited
AKR1B2 more potently than AKR1B19. AKR1B19 and AKR1B2 were
also inhibited by sulindac, quercetin and diethylstilbestrol.
Although its inhibitory potency was low, diphenic acid, an alde-
hyde reductase inhibitor [38], inhibited only AKR1B19. It should
be noted that another aldehyde reductase inhibitor, valproate,
did not affect the activities of both AKR1B19 and AKR1B2.
lM substrate for
a
a
a
a
a
a
diol (3b), (a) the control cells, and (b) the overexpressed cells. (B) Reduction of 5b-
dihydrotestosterone (5b-DHT). Chromatograms: (a) the control cells, (b) the
overexpressed cells, and (c) the overexpressed cells cultured with 2
lM HAHE.
The control cells produced 5b-androstane-3 ,17b-diol (3 ), whereas the overex-
a
a
pressed cells showed the formation of large amounts of 5b-androstane-3b,17b-diol
(3b), which was significantly decreased by the addition of the AKR1B19 inhibitor,
HAHE. The peaks other than the above reduced products and substrates are due to
unknown substances (⁄).
Table 3
Comparison of inhibitor sensitivity between AKR1B19 and AKR1B2.
Inhibitora
IC50
(
lM)
Ratio 1B2/1B19
and prostaglandins (D2, E2, and A1). In the reverse reaction, the
AKR1B19
AKR1B2
enzyme oxidized 3b-hydroxy-5
significant dehydrogenase activity for
(dehydroepiandrosterone, pregnenolone, and 5-pregnene-3b,20
diol) and 3 -hydroxysteroids (5 /b-androstan-3 -ol-17-ones, 5
b-androstane-3 ,17b-diols, /b-pregnan-3 -ol-20-ones and
lithocholic acid). The kcat/Km values for the 3b-hydroxysteroids
were higher than those for geranylgeraniol and farnesol, which
were most efficiently oxidized by AKR1B19 among non-steroidal
substrates including 4-oxo-2-nonenol, 4-hydroxy-2-nonenol, and
benzyl alcohol (Table S3, Supplementary Data). NADP+ was a pref-
a
/b-dihydrosteroids, but show no
AKR1B10 inhibitors
PHPC
HAHE
Oleanolic acid
Bisdemethoxycurcumin
D
5-3b-hydroxysteroids
0.008
0.060
0.40
0.011
5.9
15
1.3
98
38
a
-
a
a
a
a
a/
0.60
0.51
0.9
a
5
a
AR inhibitors
Minalrestat
Tolrestat
Zopolrestat
Epalrestat
AL1567
0.043
0.16
1.2
1.5
1.7
0.023
0.016
0.27
0.68
1.8
0.5
0.1
0.2
0.5
1
Sorbinil
5.3
2.1
0.4
erable coenzyme (Km = 1.3
40
lM), and the dehydrogenase activity for
M 5b-androstan-3b-ol-17-one with 1 mM NAD+ was only 8.7%
Other inhibitors
Sulindac
Quercetin
Diethylstilbestrol
Diphenic acid
l
of the NADP+-linked activity. It should be noted that no activities of
3-ketosteroid reductase and 3b-HSD were observed for AKR1B2
and human AKR1B10.
0.86
1.5
2.8
31
36
0.1
0.9
>8
0.17
2.4
260
>2000b
As AKR1B19 specifically oxidized 3b-hydroxysteroids, the re-
duced products of 5b-pregnane-21-ol-3,20-dione, dehydrolitho-
cholic acid, 5b-dihydrotestosterone (androstan-17b-ol-3-one),
5b-androstane-3,17-dione and 5b-pregnane-3,20-dione were
identified as 5b-pregnane-21,3b-diol-20-one, isolithocholic acid,
a
Abbreviations: PHPC, (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-
yl)-2H-chromene-3-carboxamide; HAHE, 3-(4-hydroxy-2-methoxyphenyl)acrylic
acid 3-(3-hydroxyphenyl)propyl ester; and AL1567, DL-spiro(2-fluoro-9H-fluoren-
9,40-imidazolidine)-20,50-dione.
b
No significant inhibition at 2 mM.

28
S. Endo et al. / Archives of Biochemistry and Biophysics 527 (2012) 23–30
Table 4
Inhibition patterns and constants of AKR1B19 inhibitors.
a
b
Inhibitor
Inhibition pattern
Inhibition constant (nM)
Kis
Kii
Reduction reaction
PHPC
HAHE
Noncompetitive
Uncompetitive
6.7 0.4
7.8 0.9
40
4
Oxidation reaction
PHPC
HAHE
Oleanolic acid
Minalrestat
Tolrestat
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
2.7 0.4
26
97
16
78
5
5
2
5
Diphenic acid
6600 900
a
Abbreviations of PHPC and HAHE are described in Table 3.
The varied substrates are pyridine-3-aldehyde and 5b-androstan-3b-ol-17-one
b
in the reduction and oxidation, respectively.
Fig. 4. Elution patterns of the activities of lung NADP+-linked 3b-HSD and NADPH-
linked aldehyde reductases on Q-Sepharose chromatography. After the enzyme
fraction purified by the Sephadex G-100 filtration of male rabbit lung cytosolic
proteins was applied, the adsorbed proteins (ꢂ ꢂ ꢂ) were eluted with a linear gradient
of 0–0.1 M NaCl (?). Activities: 3b-HSD towards 40
lM 5b-androstan-3b-ol-17-one
(s), pyridine-3-aldehyde reductase ( ), and
D
D-glucuronate reductase (j).
3b-HSD activity was eluted only in the non-adsorbed fractions,
whereas the pyridine-3-aldehyde reductase activity separated into
two peaks, one of which was co-eluted with the 3b-HSD activity.
The enzyme in the non-adsorbed fractions showed low Km values
for pyridine-3-aldehyde, 5b-dihydrotestosterone and 5b-andro-
stan-3b-ol-17-one (10, 6.2 and 10
ity to HAHE and diphenic acid (the IC50 values are 0.095 and
430 M, respectively), which are similar to those of recombinant
AKR1B19 (Tables 1 and 2). On the other hand, the enzyme eluted
in the NaCl gradient reduced pyridine-3-aldehyde (Km = 0.6 mM)
lM, respectively), and sensitiv-
Fig. 3. RT-PCR analysis for expression of mRNAs for AKR1B19, AKR1B2 and AKR1D3
in rabbit tissues. Tissues: brain (B), lung (Lu), heart (H), stomach (S), liver (Li), renal
cortex (Rc), renal medulla (Rm), adrenal gland (A), small intestine (I), colon (C), and
testis (T). The expression of mRNA for b-actin is shown as the control.
l
and
reductase activity towards 5b-dihydrotestosterone and
The -glucuronate activity was inhibited by both diphenic acid
and valproate (IC50 values are 182 and 171 M, respectively). The
properties of the adsorbed enzyme exhibiting pyridine-3-aldehyde
reductase activity are similar to those of aldehyde reductase
[38,39].
D
-glucuronate (Km = 2.9 mM) at similar rates, but showed no
D
-glucose.
In the reduction of pyridine-3-aldehyde by AKR1B19, the inhibi-
tion patterns of PHPC and HAHE were noncompetitive and uncom-
petitive, respectively, with respect to the substrate (Table 4). In the
reverse reaction of 5b-androstan-3b-ol-17-one oxidation, the inhi-
bition patterns of PHPC and HAHE were competitive with respect
to the substrate. Other inhibitors, minalrestat, tolrestat, oleanolic
acid and diphenic acid, were all competitive inhibitors with respect
to the alcohol substrate.
D
l
Alteration of reactivity for steroids by mutagenesis
Tissue distribution
The outstanding difference of AKR1B19 from human and rodent
AR-like proteins was the efficient and stereo-specific reduction of
3-keto-5b-dihydrosteroids, despite of their high SI, as described
above. Among the substrate-binding residues predicted from the
crystal structure of AKR1B10 and site-directed mutagenesis [40],
two residues at positions 303 and 304, located in a region known
as the C-terminal substrate-binding loop, are different between
the human and rabbit enzymes (Fig. S1, Supplementary Data). The
corresponding residues are Phe and Met in AKR1B19, whereas they
are Gln and Ser in AKR1B10. In addition, Leu116 that corresponds to
a substrate-pocket residue of the AKR superfamily proteins [41] is
replaced to Phe in AKR1B10. To examine whether the three residues
are responsible for the reduction of 3-ketosteroids, we prepared
three single mutant AKR1B19s (L116F, F303Q and M304S) and a
double F303Q/M304S mutant enzyme, and compared the effects
of the mutations on kinetic constants for 3-ketosteroids (5b-preg-
nane-21-ol-3,20-dione and 5b-dihydrotestosterone) and non-ste-
roidal substrates (methylglyoxal and pyridine-3-aldehyde). The
mutation of L116F did not apparently affect the kinetic constants,
whereas those of F303Q and M304S resulted in significant altera-
tions of Km and kcat/Km values for the steroidal substrates (Table 5).
The tissue distribution of AKR1B19 was accessed by RT-PCR,
and compared with that of AKR1B2 (Fig. 3). The mRNAs for the
two enzymes were detected in many rabbit tissues, of which the
adrenal gland showed high expression of the two mRNAs. Since
5b-dihydro-C19/C21-steroids that were better substrates for
AKR1B19 than their 5
by
4-3-ketosteroid 5b-reductase, the expression of the mRNA for
rabbit
4-3-ketosteroid 5b-reductase (AKR1D3) in rabbit tissues
a-isomers (Table 1) are metabolically formed
D
D
was also examined by RT-PCR. This mRNA was detected in all rab-
bit tissues, although its expression levels in the renal cortex, adre-
nal gland, small intestine and colon were high.
To examine the expression of AKR1B19 protein and its identity
with cytosolic 3b-HSD in rabbit tissues, the NADP+-linked 5b-
androstan-3b-ol-17-one dehydrogenase and NADPH-linked pyri-
dine-3-aldehyde reductase activities were partially purified from
the lung, from which the cDNA for the enzyme was isolated. The
two enzyme activities were eluted at the same low-molecular
weight (around 35-kDa) fraction on the Sephadex G-100 filtration,
but separated on the Q-Sepharose chromatography (Fig. 4). The

S. Endo et al. / Archives of Biochemistry and Biophysics 527 (2012) 23–30
29
Table 5
over AKR1B1 [23,35]. Compared to human AKR1B10 [43,45], the
reactivity towards all-trans-retinal of AKR1B19 is low, but, instead,
that towards ONE is significantly high. The catalytic efficiency for
ONE of AKR1B19 is the highest among those reported with other
AR-like proteins (AKR1B7 [46], AKR1B14 [19] and AKR1B18 [20]),
and ARs (AKR1B1 [27] and AKR1B2). AKR1B19 also reduced GS-
ONE that is formed by nonenzymatic reaction and glutathione-S-
transferase [27,47], and its kcat/Km values are about an order mag-
nitude higher than those of AKR1B1 [27], AKR1B14 [19], and
Alteration of kinetic constants for steroidal and non-steroidal substrates by
mutagenesis.
Substrate
Enzyme Km
M)
kcat
kcat/Km
(
l
Mu/ (minꢀ1
WTa
)
Mu/
WTa
(l
Mꢀ1 minꢀ1) Mu/
WTa
5b-Pregnane-21- WT
ol-3,20-dione L116F
F303Q
2.4 0.2
2.3 0.1
13 1.0
1
1
5
5.5 0.5 1
3.8 0.1 0.7 1.7
3.7 0.3 0.5 0.25
2.3
1
0.7
0.1
0.05
0.01
M304S
F303Q/
23
50
2
5
10 2.5 0.3 0.4 0.11
21 1.4 0.1 0.2 0.028
AKR1B18 [20]. In addition, this enzyme reduced other a,b-unsatu-
rated aldehydes derived from lipid-peroxidation [29,48,49]. The
substrate specificity, together with the ubiquitous tissue distribu-
tion, suggests that AKR1B19 is implicated in detoxification of the
reactive aldehydes, especially the most highly reactive lipid-de-
rived aldehyde, ONE.
AKR1B19 also reduced isocaproaldehyde, which is the product
of side-chain cleavage of cholesterol in the steroidogenesis of
endocrine glands. Previously, three forms of 36-kDa NADPH-
dependent isocaproaldehyde reductase were isolated from rabbit
adrenal gland [25]. Although the two of the three isocaproaldehyde
reductases are identified as ARs, the nature of the third enzyme
form remains unknown. This third enzyme form shows an acidic
M304S
5b-Dihydro-
WT
3.5 0.2
7.2 2.0
1
2
4
6
6.8 0.7 1
6.4 2.2 1
4.8 0.3 0.7 0.28
2.8 0.1 0.4 0.14
1.9
0.88
1
testosterone L116F
F303Q
0.5
0.1
0.07
0.02
17
20
39
2
1
3
M304S
F303Q/
M304S
11 1.2 0.2 0.2 0.031
Methylglyoxal
WT
L116F
F303Q 253 10
M304S 209 20
F303Q/ 250 14
M304S
222 25
180 30
1
1
1
1
1
18 0.2 1
0.080
0.11
0.13
0.10
0.10
1
1
2
1
1
19
35 10
22
24
3
1
2
1
2
2
1
Pyridine-3-
aldehyde
WT
11
22
11
39
48
2
2
1
3
6
1
2
1
4
4
43
36
52
47
4
6
7
6
1
1
1
1
2
4.1
1.7
4.7
1.2
1.5
1
0.4
1
0.3
0.4
pH optimum of 6.3, inability to reduce D-glucose, low sensitivity
L116F
F303Q
M304S
F303Q/
M304S
to tolrestat, AL1567 and sorbinil, and moderate inhibition by
diphenic acid. The properties are similar to those of AKR1B19,
whose mRNA was highly expressed in the adrenal gland. Therefore,
AKR1B19 may be identical to the third form of adrenal isocaproal-
dehyde reductase previously isolated.
72 10
a
Ratio of mutant enzyme to WT.
The most striking difference of AKR1B19 from human and ro-
dent AR and AR-like proteins is its ability to efficiently reduce 3-
ketosteroids into 3b-hydroxysteroids. Among mammalian ARs
and AR-like proteins, bovine AR (AKR1B5) [50] and Chinese-ham-
The double mutation of F303Q/M304S further impaired the affinity
and catalytic efficiency, although it did not affect the stereospecific
reduction of the two 3-ketosteroids into the corresponding 3b-
hydroxysteroids (data not shown). In contrast, the mutations did
not affect the kinetic constants for the small non-steroidal sub-
strates, except that the M304S mutation caused a small (fourfold)
increase in the Km value for pyridine-3-aldehyde.
ster AKR1B9 [51] reduce the 20-keto group of 17
terone, and human AKR1B10 exhibits low 20 -HSD activity [17].
Although AKR1B7 exhibits low reductase activity for 3-keto bile
acids [16], 3-keto-5 /b-dihydro-C19/C21-steroids are not reduced
a-hydroxyproges-
a
a
by human AKR1B10 and AKR1B1 [17], rat AKRs (1B13, 1B14 and
1B18) [18–20], mouse AKR1B7 [46, unpublished data] and rabbit
AKR1B2 [this study]. Bile acids and 3-ketosteroids are inhibitors
for human AKR1B10 and AKR1B1 [17], and bile acids, other than
dehydrocholic acid, are activators for AKR1B14 [46]. Thus,
AKR1B19 is the first AKR1B subfamily enzyme that exhibits signif-
Discussion
In the present study, for the first time we have cloned the full-
length cDNA for AKR1B19, and characterized AKR1B19 as a func-
tional NADPH-dependent AKR that reduces a variety of aldehydes
icant reductive 3b-HSD activity towards 3-keto-5a/b-
dihydrosteroids.
and
a
-dicarbonyl compounds, and some xenobiotic p-quinones
The kcat/Km values for the 3-ketosteroid substrates of AKR1B19
are comparable or superior to those of human AKR1C1 [4], 17b-
HSD type-7 [5] and DHRS4 [6]. In addition, AKR1B19 reduces a
large number of 3-ketosteroids compared to these human en-
and ketones. AKR1B19 shows more than 80% amino acid SI not
only with human AR-like proteins (SI) [AKR1B10 (86%) and
AKR1B15 (82%)], but also with other animal AR-like proteins (SI):
mouse AKRs [1B8 (85%) and 1B7 (80%)]; rat AKRs [1B13 (84%),
1B14 (81%) and 1B18 (81%)]; and Chinese hamster AKR1B9 (85%).
Although the enzymatic properties of human AKR1B15 have not
been characterized well [42], the broad substrate specificity for
aldehydes is common among the other AR-like proteins. However,
the enzymes differ in other properties. For examples, human
AKR1B10 is distinct from the rodent AR-like proteins, in its great
ability to reduce all-trans-retinal [43], isoprenyl aldehydes (farne-
sal and geranylgeranial) [17] and some p-quinones (TBE and 2-tert-
butyl-1,4-benzoquinone) [44]. Rat AKR1B18 is distinct from the
other rodent and human AR-like proteins, especially in its efficient
reduction of ONE and activation by organic acids [20].
zymes. The metabolism of 5a- and 5b-dihydrotestosterones in
the AKR1B19-overexpressed cells supports that the enzyme cata-
lyzes the strict 3b-stereoselective reduction in vivo. Currently,
there are no reports on rabbit enzymes with the reductive 3b-
HSD activity, as described in the Introduction section. AKR1B19
mRNA was expressed in many rabbit tissues, and the identity of
AKR1B19 with cytosolic 3b-HSD was evidenced by the co-purifica-
tion of the two enzyme activities from rabbit lung. AKR1B19 may
play a role in the intracellular pre-receptor formation of the active
3b-hydroxyandrostanes and 3b-hydroxypregnanes [8–13], as well
as in the inactivation of the potent androgen 5
one. The enzyme did not reduce
4-unsaturated steroids, but re-
duced various 5 - and 5b-dihydrosteroids that are formed from
testosterone, progesterone and corticoids by steroid reductases.
Considering the ubiquitous expression of the mRNA for
4-steroid
a-dihydrotestoster-
D
AKR1B19 mostly resembles human AKR1B10 among the AR-like
proteins, in its ability to reduce all-trans-retinal and efficient
reduction of the isoprenyl aldehydes and p-quinones. In addition,
AKR1B19 was inhibited by the human AKR1B10 inhibitors, of
which HAHE and oleanolic acid showed high selectivity to
AKR1B19 over AKR1B2, which is similar to that of human AKR1B10
a
D
5b-reductase (AKR1D3) in rabbit tissues, AKR1B19 in concert with
AKR1D3 also contributes to catabolic pathways of these steroid
hormones. Thus, the enzyme is important for the metabolism of

30
S. Endo et al. / Archives of Biochemistry and Biophysics 527 (2012) 23–30
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endogenous steroids and cytotoxic aldehydes rather than that of
xenobiotic carbonyl compounds.
The results of the mutagenesis of F303Q/M304S have revealed
that both Phe303 and Met304 play critical roles in recognition of
3-ketosteroid substrates by AKR1B19, although other residues are
probably involved in the steroid binding because of lack of com-
plete abolishment of reductive 3b-HSD activity by the mutagene-
sis. The two residues are present in a so-called C-terminal loop of
the tertiary structures of enzymes of the AKR superfamily, in which
amino acid difference in this and other two loop regions between
different enzymes has been suggested to determine the substrate
specificity of the enzymes [2,41]. In the crystal structures of HSDs
belonging to the AKR1C subfamily, apolar residues (at positions
306, 308 and 310) in the C-terminal loop interact the steroidal sub-
strates or inhibitors [15,41]. The C-terminal loop of AKR1B1 is crit-
ical for catalytic efficiency and substrate specificity [52], and a role
of Cys304, a C-terminal loop residue of AKR1B10, in binding of the
cyclohexene ring of all-trans-retinal has been shown by crystallo-
graphic and site-directed mutagenesis studies [40,43]. Based on
the present mutations and the crystal structure of AKR1B10, the
binding mode of the 3-ketosteroids in AKR1B19 can be speculated
as follows. The 3-keto group on the A ring of the steroid must be
close to the catalytic residues of Tyr48 and His111, so that the
opposite side of this steroid molecule may orientate towards
hydrophobic residues, Phe303 and Met304 to form hydrophobic
interactions. The formation of this hydrophobic interaction is sup-
ported by no significant effects of the F303Q/M304S mutation on
the kinetic constants for the smaller and more hydrophilic sub-
strates, methylglyoxal and pyridine-3-aldehyde. Structural studies
using molecular modeling techniques and crystallization of
AKR1B19 complexed with the coenzyme and steroid are now in
progress in order to clarify the steroid recognition of the rabbit en-
zyme leading to efficient 3b-HSD activity.
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Acknowledgments
This work was partly founded by Grant-in-aid for Young Scien-
tists (B) and Scientific Research (C) from the Japan Society for the
Promotion of Science, and by a Sasakawa Scientific Research Grant
from Japan Science Society.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.abb.2012.07.012.
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