Mouse AKR1E1 is an ortholog of pig liver NADPH dependent 1,5-anhydro-D-fructose reductase

Article abstract of DOI:10.1271/bbb.70612

In many organisms, glycogen gives rise to 1,5-anhydro-D-fructose (AF), which is reduced to 1,5-anhydro-D-glucitol (AG). AF reductase, which catalyzes the latter reaction, was purified from pig liver, but mouse ortholog has not yet been reported. In the database, aldo-keto reductase family 1, member E1 (AKR1E1) showed highest homology to pig enzyme. We confirmed that cloned AKR1E1 is mouse ortholog based on enzymatic properties of purified recombinant protein.

Full text of DOI:10.1271/bbb.70612

Biosci. Biotechnol. Biochem., 72 (3), 872–876, 2008
Note
Mouse AKR1E1 Is an Ortholog of Pig Liver NADPH Dependent
1,5-Anhydro-D-Fructose Reductase
y
Motoki SAKUMA and Shunichiro KUBOTA
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo,
3
-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan
Received September 26, 2007; Accepted December 25, 2007; Online Publication, March 7, 2008
[doi:10.1271/bbb.70612]
In many organisms, glycogen gives rise to 1,5-
drastically. Hence, the blood AG level is used as clinical
marker of diabetes mellitus.
The mammalian cell membrane has high permeability
anhydro-D-fructose (AF), which is reduced to 1,5-
anhydro-D-glucitol (AG). AF reductase, which catalyzes
the latter reaction, was purified from pig liver, but
mouse ortholog has not yet been reported. In the
database, aldo-keto reductase family 1, member E1
4
)
to AG, comparable to glucose. But although AG is
analogous to glucose, it is suggested that it is transported
not by the glucose transporter, but by an AG specific
4
)
(
AKR1E1) showed highest homology to pig enzyme. We
transporter. The existence of an AG maintaining
system implies that AG has an important physiological
function.
confirmed that cloned AKR1E1 is mouse ortholog based
on enzymatic properties of purified recombinant pro-
tein.
Shiga et al. reported that Esherichia coli C600
steadily degrades about 1/1,000 portion of accumulated
glycogen to AF, and that AF is reduced to AG when the
Key words: glycogen degradation; 1,5-anhydro-D-fruc-
tose; 1,5-anhydro-D-fructose reductase; 1,5-
anhydro-D-gucitol; anhydrofructose path-
way
2
)
glucose in the medium is exhausted. They also reported
that AG in the medium accelerates glycogen utiliza-
2
)
tion. Based on these findings, they suggested that the
glycogen metabolism controls the AF pathway, and that
2
)
1
,5-anhydro-D-fructose (AF) in organisms was first
AG is an extracellular signaling molecule in bacteria.
The AF concentration and the glycogen contents in
reported in fungi. Later, Yu et al. purified and
characterized the enzyme which catalyzes the degrada-
tion of ꢀ-1,4-glucan (such as glycogen and starch) into
3
)
various rat organs are positively correlated. Human
K562 erythroleukemia cells reduce AF in culture
1
)
5)
AF from red algae and they termed this the AF
pathway. After that, AF pathway was reported in wide
medium, but the reducing activity is strongly sup-
5
)
pressed by a physiological level of glucose. Hence,
a relation between glycogen metabolism and the AF
pathway is probable also in mammalian cells.
2
)
variety of organisms such as bacteria, fungi, higher
3
)
plants and mammals.
In some organisms, AF is catabolized to antimicro-
bials, an antioxidant, an anti-freezing agent, or 1,5-
anhydro-D-mannitol. But in most organisms, it is
Based on partial amino acid sequences obtained from
6
)
pig liver AF reductase, we searched for the putative
mouse AF reductase gene by BLAST search (tblastn).
The result showed that AKR1E1 (aldo-keto reductase
family 1, member E1) had the highest homology in the
database (66% of the residues were identical). Bohren
et al. cloned and expressed this cDNA, and determined
2
,3)
reduced to 1,5-anhydro-D-glucitol (AG).
AG is the 1-deoxy form of glucopyranose. In human
body fluid, AG concentration is about 1/40 of glucose
level, and is the second most abundant polyol. In the
mammalian body, it is chemically highly stable; the only
known metabolic reaction is reversible phosphorylation
7
)
some properties of the recombinant protein. Azuma
et al. cloned and expressed human testis specific
8
)
of the C hydroxyl group. Despite its metabolic inert-
6
ortholog. But neither group determined the physiolog-
ical function of this enzyme.
ness, the AG level in body fluid is definitely controlled
by certain mechanisms. It is not influenced by hormones,
food intake, or starvation. But when the hyperglycemic
state is continued, AG is excreted in the urine together
with glucose, and the blood AG level is reduced
As shown in Fig. 1A, mouse aldose reductase also has
high homology to AKR1E1 (61% of the residues were
identical). But, as shown in Fig. 1B, most of residues
9
)
which constitute the substrate binding pocket were
y
To whom correspondence should be addressed. Tel/Fax: +81-3-5454-6869; E-mail: kubota@idaten.c.u-tokyo.ac.jp
Abbreviations: AF, 1,5-anhydro-D-fructose; AG, 1,5-anhydro-D-glucitol; AKR, aldo-keto reductase; AKR1E1, aldo-keto reductase family 1,
member E1

Determination of Mouse AF Reductase
873
A
B
Protein
Residue
48 50 51 80 111 112 114 122 123 220 299 301 303
Mouse aldose
Reductase
AKR1E1
Pig AF
A
A
A
V
L
L
Y
Y
Y
W
W
H
H
H
W
W
W
T
Y
D
D
F
I
W
--
C
A
S
L
F
F
S
T
I
M
M
n.d.
L
--
Reductase
Fig. 1. Amino Acid Sequence Alignment of AKR1E1, Pig AF Reductase and Mouse Aldose Reductase.
A, Sequences of mouse aldose reductase were obtained from the NCBI database. Alignment was performed using the program
blastp. Residues of pig AF reductase and mouse aldose reductase that are identical to AKR1E1 are shaded. Framed residues are residues
that form B-loop. B, Substrate binding pocket residues of mouse aldose reductase are aligned with the corresponding residues of AKR1E1
and pig AF reductase. Residues in the gray box were residues identical or similar to mouse aldose reductase. Residues in the black box
were residues identical or similar only as between AKR1E1 and pig AF reductase. n.d., not determined. - -, corresponding residue does
not exist.
identical or similar as between AKR1E1 and pig AF
reductase, but about half of the pocket residues were
different as between AKR1E1 and aldose reductase.
In addition, complemental parts for aldose reductase’s
Mouse liver total RNA (from C57BL/6J strain, 8-
week-old male) was used as a starting material for
cloning of the AKR1E1 gene. First strand cDNA
synthesis was performed using a Superscript II reverse
transcription kit (Invitrogen, Carlsbad, CA) using
oligo (dT) as a primer. Subsequent PCR was carried
2
14th to 219th amino acid residues were deleted both
in pig AF reductase and AKR1E1 (Fig. 1A). These
residues were part of the aldo-keto reductase family’s
common motif called the B-loop, which is thought to be
out using ExTaq polymerase (Takara Bio, Ohsu, Japan),
0
a
GAAAACATCCC-3 ; the NcoI site underlined) and a
forward primer (5 -AATCCGGATCCACCATG-
0
9
)
related to substrate specificity. From these facts, it is
likely that AKR1E1 is AF reductase of the mouse.
0
reverse primer (5 -AAGCTCGAGTCAATACTCTAT-

874
M. SAKUMA and S. KUBOTA
A
Step
Total protein Total activities Specific activities Recovery Purification
(
mg)
(units)
2.78
(units/ mg)
0.17
(%)
100
48.9
(-fold)
1
1
2
3
. Cell lysate
. DE 52
16.1
0.94
1.36
1.44
8.3
. Red Sepharose
CL-6B
0.18
0.53
3.01
19.1
17.4
B
175
8
6
4
3
2
8
33
34 kDa
2
1
5
7
(
kDa)
1
2
3
4
Fig. 2. Purification of Recombinant Protein.
A, Summary of recombinant AKR1E1 protein purification. One unit of the enzyme was defined as the amount of the enzyme that reduces
1
mmol of AF in 1 min. B, SDS–PAGE image of purified recombinant protein. Lane 1, molecular weight marker; lane 2, cell lysate; lane 3, DE52
fraction; lane 4, Red Sepharose fraction. Electrophoresis was performed with 12% gel and the amount of protein in each lane was 10 mg. The gel
was stained with Coomassie brilliant blue R-250. The molecular weight of the recombinant protein was estimated by its mobility as compared
with a molecular weight marker.
0
GTGGAAAGG-3 ; the XhoI site is underlined). Primers
were designed to contain restriction sites for site-
directied ligation to pTriEx4 multisystem expression
vector (Novagen, Darmstadt, Germany). Amplified PCR
products were ligated to pGEM-T (Promega, Madison,
WI) TA-cloning vector, and the resulting vector was
digested in two ways by NcoI-BstXI double-digestion
and BstXI-XhoI double-digestion, because AKR1E1
has another NcoI site within the ORF. The resulting
fragments were ligated to pTriEX4 vector using DNA
ligation kit ver. 2.1 (Takara).
sonicated, and centrifuged. The collected supernatant
was diluted 3-fold with double distilled water and
applied to 15 ml of DE52 column. The column was
washed with 1 bed volume of buffer A and 0.05 M NaCl
in buffer A, and eluted with 0.5 bed volume of 0.1 M
NaCl in buffer A. The eluent was applied directly to
5 ml of Red Sepharose CL6B column. The column was
washed with 1 bed volume buffer A and 1 M NaCl
in buffer A, and eluted with 1.5 M NaCl in buffer A
(Fig. 2A). The specific activity of the purified enzyme
was 3.01 units/mg protein, higher than pig enzyme
(1.16 units/mg protein). The recombinant protein was
purified to homogeneity as judged by CBB-stained
SDS–PAGE (Fig. 2B).
For bacterial protein expression, we used E. coli
strain BL21 (DE3) pLysS as host cells. Plasmid-
harboring bacteria were grown in 50 ml of Luria-broth
containing 50 mg/ml of ampicillin at 37 C until OD
00 nm reached 0.5. Protein expression was induced by
.5 mM isopropyl-ꢁ-D-thiogalactoside. The cells were
collected by centrifugation, and the pellet was washed
with 0.25 M glucose in 20 mM sodium phosphate buffer
pH 7.0) (buffer A), and resuspended in 5 ml of buf-
fer A. The suspension was frozen and then thawed,
ꢀ
The substrate specificities of recombinant protein for
many carbonyl compounds are summarized in Fig. 3A.
The starch-derived AG-free AF we used in experiments
was a gift from Danisco A/S (Copenhagen, Denmark).
The substrate specificity profile of the recombinant
6
0
6
)
(
protein is concordant to pig enzyme. Among the
compounds we tested, AF was the most preferred

Determination of Mouse AF Reductase
875
A
Concentration
Relative activity of
recombinant protein (%)
Relative activity of
pig AF reductase (%)
Substrate
(
mM)
0.3
0.5
0.5
0.5
0.5
0.5
0.5
0.5
AF
100
n. d.
0.9
100
n. d.
n. d.
15.2
53.4
n. d.
7.3
Glucose
Fructose
Glucronic acid
,3-butanedione
DL-glycelaldehyde
Formaldehyde
Acetone
5.1
2
76.4
n. d.
1.9
n. d.
n. d.
9
,10-phenanthrene-
0
.2
18.0
n. d.
--
quinone
AG
reverse reaction)
100
n. d.
(
B
C
K
m
V
max
molecular activity
(
1
mM)
.02
(µmol / min)
(molecules / sec)
9.3
0.57
Fig. 3. Kinetic Properties of the Recombinant Protein.
A, Enzyme activity was determined by monitoring the decrease in absorbance at 340 nm due to NADPH consumption. The reaction mixture
contained 0.2 mM NADPH, indicated concentration of each substrate and appropriate amount of enzyme in 20 mM phosphate buffer (pH 7.0).
ꢀ
þ
The mixture was incubated at 37 C for 10 min. In the reverse reaction assay, 10 mM NADP was added as a cofactor and the mixture was
ꢀ
incubated at 37 C for 30 min. n.d., not detectable. - -, experiment was not performed. B, Lineweaver-Burk plot of AF reducing reaction of
recombinant protein. Reaction mixture contained 4 mM NADPH and variety of AF concentrations (0.15, 0.3, 0.6, 1.5, and 3.0 mM) and
appropriate amounts of enzyme in 20 mM phosphate buffer (pH 7.0). The enzyme activity was determined by measuring the amount of AG
product. Assays were performed in tripricate for each AF concentration. C, Kinetic values of recombinant protein to AF. Kinetic constants were
calculated using linear least-squares fitting to a Lineweaver-Burk plot.
substrate. 2,3-butanedione was a relatively good sub-
strate. Glucronic acid and formaldehyde were reduced
weakly and activity against glucose, DL-glycelaldehyde
and acetone were not detected under our experimental
conditions. The only discrepancy was that recombinant
protein has very weak activity against fructose. It is
uncertain whether the difference was derived from
species differences, or the activity of residual impurities
that preferred fructose as substrate. It has been reported
that recombinant protein expressed by AKR1E1 reduced
9,10-phaenanthrenequinone effectively.7,8) This was
confirmed in this study. Moreover, recombinant protein
þ
did not catalyze the reverse reaction, viz., NADP -
dependent AG oxidation, as was observed in the case of
6
)
pig enzyme. These enzymatic properties support the
assumption that the recombinant protein is AF reductase
of the mouse.
The high Km value and low molecular activity of the
recombinant protein was also concordant to pig liver
6
)
enzyme. The Km of the recombinant protein was

876
M. SAKUMA and S. KUBOTA
1
.02 mM, about 2.3-fold higher than that of pig enzyme
0.44 mM), but the difference is within acceptable limits.
Efficient purification and characterization from red sea-
weeds. Biochim. Biophys. Acta, 1156, 313–320 (1993).
2) Shiga, Y., Kametani, S., Kadokura, T., and Akanuma, H.,
1,5-Anhydroglucitol promotes glycogenolysis in Esche-
richia coli. J. Biochem. (Tokyo), 125, 166–172 (1999).
(
Molecular activity of recombinant protein was 9.3 s
very close to that of pig enzyme (8.7 s 1) (Fig. 3B, C).
ꢁ1
,
ꢁ
Taken together similarities such as in amino acid
sequence, substrate specificities, and kinetic properties,
it is highly likely that the recombinant protein expressed
from the AKR1E1 gene is the mouse ortholog of pig AF
reductase.
Although AG is widely used as a marker for diabetes
mellitus in the clinical field, the physiological role of
AG and its synthetic pathway are poorly understood.
For study of AG, application of molecular biological
methods should be useful. In the present study, we
identified the mouse gene for AF reductase. We intend
to elucidate the AF pathway by overexpression or
knockout of the AF reductase gene using molecular and
biological techniques.
3
4
5
6
7
)
)
)
)
)
Kametani, S., Shiga, Y., and Akanuma, H., Hepatic
production of 1,5-anhydrofructose and 1,5-anhydrogluci-
tol in rat by the third glycogenolytic pathway. Eur. J.
Biochem., 242, 832–838 (1996).
Suzuki, M., Akanuma, H., and Akanuma, Y., Transport of
1,5-anhydro-D-glucitol across plasma membranes in rat
hepatoma cells. J. Biochem. (Tokyo), 104, 956–959
(1988).
Suzuki, M., Kametani, S., Uchida, K., and Akanuma, H.,
Production of 1,5-anhydroglucitol from 1,5-anhydrofruc-
tose in erythroleukemia cells. Eur. J. Biochem., 240,
2
3–29 (1996).
Sakuma, M., Kametani, S., and Akanuma, H., Purification
and some properties of a hepatic NADPH-dependent
reductase that specifically acts on 1,5-anhydro-D-fructose.
J. Biochem. (Tokyo), 123, 189–193 (1998).
Bohren, K. M., Barski, O. A., and Gabbay, K. H.,
Characterization of a novel murine aldo-keto reductase.
Adv. Exp. Med. Biol., 414, 455–464 (1997).
Acknowledgment
This study was supported by a grant from the Ministry
of Education, Culture, Sports, Science, and Technology
of Japan.
8) Nishinaka, T., Azuma, Y., Ushijima, S., Miki, T., and
Yabe-Nishimura, C., Human testis specific protein: a new
member of aldo-keto reductase superfamily. Chem. Biol.
Interact., 143–144, 299–305 (2003).
References
9
)
Jez, J., Bennett, M., Schlegel, B., Lewis, M., and Penning,
T., Comparative anatomy of the aldo-keto reductase
superfamily. Biochem. J., 326, 625–636 (1997).
1
)
Yu, S., Kenne, L., and Pedersen, M., ꢀ-1,4-glucan lyase,
a new class of starch/glycogen degrading enzyme. I.