Recently, we have reported that human AKR1B1, mouse AKR1B3 and mouse AKR1B7 are associated with PGF2a synthase PGFS; EC 1.1.1.188 activity, which catalyzes the reduction of PGH2, a commo
Trang 1Catalytic mechanism of the primary human prostaglandin
synthase activity in the absence of NADP(H)
Nanae Nagata1, Yukiko Kusakari2, Yoshifumi Fukunishi3, Tsuyoshi Inoue2and Yoshihiro Urade1
1 Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Japan
2 Department of Materials Chemistry, Osaka University, Japan
3 Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan
Introduction
Aldo-keto reductases (AKRs) are soluble monomeric
proteins with molecular masses of 37 kDa with
NADPH-dependent oxidoreductase activity AKR
proteins are widely distributed in prokaryotes and
eukaryotes, fall into 15 families [1] and metabolize a
number of substrates, including aldehydes,
monosaccha-rides, steroids, polycyclic hydrocarbons, isoflavonoids
and prostaglandins (PGs) in the presence of NADPH [2] Aldose reductase (EC 1.1.1.21), named AKR1B1 in human and AKR1B3 in mouse, is considered to be the prototypical enzyme of the AKR superfamily In addi-tion to these conical aldose reductases, a second group, named aldose reductase-like proteins, has been charac-terized on the basis of sequence homology (at least
Keywords
aldo-keto reductase; His; prostaglandin D2
synthase; prostaglandin F 2a synthase;
prostaglandin H2
Correspondence
Y Urade, Department of Molecular
Behavioral Biology, Osaka Bioscience
Institute, 6-2-4 Furuedai, Suita,
Osaka 565-0874, Japan
Fax: +81 6 6872 2841
Tel: +81 6 6872 4851
E-mail: uradey@obi.or.jp
(Received 21 October 2010, revised 1
February 2011, accepted 7 February 2011)
doi:10.1111/j.1742-4658.2011.08049.x
Aldo-keto reductase 1B1 and 1B3 (AKR1B1 and AKR1B3) are the pri-mary human and mouse prostaglandin F2a(PGF2a) synthases, respectively, which catalyze the NADPH-dependent reduction of PGH2, a common intermediate of various prostanoids, to form PGF2a In this study, we found that AKR1B1 and AKR1B3, but not AKR1B7 and AKR1C3, also catalyzed the isomerization of PGH2 to PGD2 in the absence of NADPH
or NADP+ Both PGD2 and PGF2a synthase activities of AKR1B1 and AKR1B3 completely disappeared in the presence of NADP+or after heat treatment of these enzymes at 100C for 5 min The Km, Vmax, pK and optimum pH values of the PGD2 synthase activities of AKR1B1 and AKR1B3 were 23 and 18 lM, 151 and 57 nmolÆmin)1Æ(mg protein))1, 7.9 and 7.6, and pH 8.5 for both AKRs, respectively, and those of PGF2a syn-thase activity were 29 and 33 lM, 169 and 240 nmolÆmin)1Æ(mg protein))1, 6.2 and 5.4, and pH 5.5 and pH 5.0, respectively, in the presence of 0.5 mM NADPH Site-directed mutagenesis of the catalytic tetrad of AKR1B1, composed of Tyr, Lys, His and Asp, revealed that the triad of Asp43, Lys77 and His110, but not Tyr48, acts as a proton donor in most AKR activities, and is crucial for PGD2 and PGF2a synthase activities These results, together with molecular docking simulation of PGH2 to the crystal-lographic structure of AKR1B1, indicate that His110 acts as a base in con-cert with Asp43 and Lys77 and as an acid to generate PGD2and PGF2ain the absence of NADPH or NADP+ and in the presence of NADPH, respectively
Abbreviations
AKR, aldo-keto reductase; PG, prostaglandin; PGDS, prostaglandin D synthase; PGFS, prostaglandin F 2a synthase; TLC, thin-layer
chromatography.
Trang 260–70% identity with aldose reductase) AKR1B7,
initially characterized as a mouse vas deferens
andro-gen-dependent protein, belongs to the aldose
reductase-like proteins X-Ray crystallographic structures of
members of the AKR superfamily have shown these
enzymes to have a common three-dimensional fold,
known as the (a⁄ b)8-barrel fold [3–6] The nucleotide
cofactor binds in an extended conformation at the top
of the a⁄ b-barrel, with the nicotinamide ring projecting
down into the center of the barrel and pyrophosphate
straddling the barrel lip [7] Kubiseski et al [8] have
established that the enzyme follows a sequential ordered
mechanism in which NADPH binds before the aldehyde
substrate and NADP+ is released after the alcohol
product is formed When, in 1992, the first complete
crystal structure of human AKR1B1 was solved, the
conserved Tyr48 was shown to fulfill the role of a
catalytic acid for NADPH-dependent reduction [5]
Recently, we have reported that human AKR1B1,
mouse AKR1B3 and mouse AKR1B7 are associated
with PGF2a synthase (PGFS; EC 1.1.1.188) activity,
which catalyzes the reduction of PGH2, a common
intermediate of various prostanoids of the two series,
to PGF2a [9] PGF2a plays a variety of physiological
roles in the body, such as the contraction of uterus,
bronchial, vascular and arterial smooth muscles [10],
regulation of pressure in the eye [11], renal filtration
[12], stimulation of hair growth [13] and regulation of
the ovarian cycle through the induction of luteolysis
[14] More recently, human AKR1B1 and mouse
AKR1B3 were identified to be the primary PGFS
[15,16] Three different pathways have been reported
for PGF2a production [10], i.e 9,11-endoperoxide
reduction of PGH2, 9-ketoreduction of PGE2 and
11-ketoreduction of PGD2, although the latter results
in the production of a PGF2a stereoisomer,
9a,11b-PGF2, not PGF2a [17] PGFS was first isolated from
mammals as an enzyme that catalyzes the reduction of
PGH2 to PGF2a, and of PGD2 to 9a,11b-PGF2 [18]
The first identified mammalian PGFS belongs to the
AKR1C family [19,20], and protozoan PGFS to the
AKR5A subfamily [21,22] PGF ethanolamide
syn-thase, which belongs to the thioredoxin-like
superfam-ily, has also recently been found to convert PGH2 to
PGF2a[23]
In this study, we introduced site-directed
mutagene-sis into the catalytic tetrad of AKR1B1, and found
that His110, not Tyr48, was crucial for PGFS activity
in the presence of NADPH Furthermore, we found
that AKR1B1 and AKR1B3, but not AKR1B7 and
AKR1C3, also catalyzed the isomerization of PGH2
to PGD2 in the absence of NADPH or NADP+ In
combination with the mutagenesis analyses and pH
titration studies, we found that His110 acted as a base
to generate PGD2 in the absence of NADPH or NADP+and as an acid to generate PGF2ain the pres-ence of NADPH Thus, this is the first report demon-strating the proton donor⁄ acceptor function of His110 during the conversion of PGH2catalyzed by AKR1B1
Results
Formation of PGF2aand PGD2from PGH2by AKR1B1
Recombinant human AKR1B1, mouse AKR1B3, mouse AKR1B7 and human AKR1C3 were expressed
in Escherichia coli and purified to be a single band as judged by SDS⁄ PAGE We incubated these purified AKR proteins with 5 lm [1-14C]PGH2 in the presence
or absence of 0.5 mm NADPH or NADP+ and ana-lyzed the reaction products by thin-layer chromatogra-phy (TLC) AKR1B1 catalyzed the reduction of the 9,11-endoperoxide group of PGH2 to produce PGF2a
in the presence of NADPH, which was defined as the PGFS activity, and the isomerization of PGH2 to PGD2 in the absence of NADPH or NADP+, which was defined as the PGD2 synthase (PGDS) activity (Fig 1A) Both PGDS and PGFS activities were not found in the presence of NADP+at all and were com-pletely inactivated by heat treatment of AKR1B1 at
100C for 5 min The PGFS and PGDS activities catalyzed by AKR1B1 were calculated to be 2.4 and 3.7 nmolÆmin)1Æ(mg protein))1, respectively (Fig 1B) AKR1B3 with 85.8% identity of the amino acid sequence of AKR1B1 also catalyzed both PGFS activ-ity [3.6 nmolÆmin)1Æ(mg protein))1] in the presence of NADPH and PGDS activity [3.3 nmolÆmin)1Æ(mg pro-tein))1] in the absence of NADPH or NADP+ How-ever, AKR1B7 (71.2% and 69.6% identity with AKR1B1 and AKR1B3, respectively) and AKR1C3 (47.4% and 47.1% identity with AKR1B1 and AKR1B3, respectively) did not catalyze PGDS activity, although these AKRs showed PGFS activity [3.9 and 0.9 nmolÆmin)1Æ(mg protein))1, respectively] in the presence of NADPH These results suggest that PGDS activity is selective to AKR1B1 and AKR1B3 among these mammalian AKR proteins
Kinetic analysis of the PGFS and PGDS activities
of AKR1B1 Figure 2A shows the pH–rate profiles of AKR1B1 for PGFS and PGDS activities The PGFS activity decreased with increasing pH, with an optimum of
pH 5.5 The pKbvalue of PGFS activity was calculated
Trang 3to be 6.19 ± 0.05 by nonlinear fitting of Eqn (1) (see
Materials and methods section) to the pH–rate profile
data However, the PGDS activity of AKR1B1
increased with increasing pH, with an optimum of
pH 8.5 The pKavalue of PGDS activity was calculated
by Eqn (1) to be 7.94 ± 0.07 Nonenzymatic
autode-gradation of PGH2 was almost constant in a range
from pH 4 to pH 9 and increased at alkaline pH
val-ues, especially at pH > 11 [24], suggesting that the pKa
value of C11 may be higher than pH 9 As PGH2does
not ionize in the pH range examined, the pH profiles of
the reaction velocity reflect the pH-dependent
ioniza-tion of the catalytic residue for the PGFS and PGDS
activities of AKR1B1 AKR1B3 also showed similar
pH–rate profiles to AKR1B1 for PGFS and PGDS
activities, although the PGDS activity of AKR1B3 was 24% of the PGFS activity (Fig S1A) The optimum
pH values were found to be pH 5.0 for PGFS activity and pH 8.5 for PGDS activity of AKR1B3 The pKb value of PGFS activity and the pKa value of PGDS activity were calculated by Eqn (1) to be 5.39 ± 0.09 and 7.57 ± 0.04, respectively, by nonlinear fitting of Eqn (1) to the pH–rate profile data
PGDS and PGFS activities of AKR1B1 were char-acterized at their optimum pH values (pH 5.5 for PGFS and pH 8.5 for PGDS) by Michaelis–Menten kinetics (Fig 2B) AKR1B1 exhibited a Km value for PGH2 of 29 lm and a Vmax value of 169 nmolÆ min)1Æ(mg protein))1for PGFS activity in the presence
of NADPH at pH 5.5, and values of 23 lm and
Fig 2 Kinetic analysis of PGFS and PGDS activities of wild-type AKR1B1 (A) V value versus pH for PGH2conversion catalyzed by wild-type AKR1B1 Wild-type AKR1B1 was incubated with 5 l M 1-[ 14 C]PGH2 in the presence of 0.5 m M NADPH for PGFS activity (s) or in the absence of NADPH or NADP+for PGDS activity (d) at 37 C for 1 min PGH 2 conversion to PGD 2 or PGF 2a was analyzed by TLC and autora-diography The corresponding values of the spot densities were plotted (B) Michaelis–Menten plots of PGFS (s) and PGDS (d) activities for AKR1B1 on PGH2at their optimum pH Wild-type AKR1B1 protein was incubated with various concentrations of PGH2in the presence of 0.5 m M NADPH for PGFS activity or in the absence of NADPH or NADP+for PGDS activity For PGDS activity, the enzymes were incubated
at 37 C for 1 min with 5 l M 1-[ 14 C]PGH2in the absence of NADPH or NADP + at pH 8.5, and, for PGFS activity, in the presence of 0.5 m M NADPH at pH 5.5.
Fig 1 PGDS and PGFS activities of recombinant AKR1B1 protein (A) Autoradiogram of TLC after incubation of AKR1B1 (each 10 lg pro-tein) with 5 l M 1-[ 14 C]PGH2in the presence or absence of cofactor at 37 C for 2 min at pH 7.0 with or without heat treatment at 100 C for 5 min (B) Enzyme activities of wild-type AKR1B1, AKR1B3, AKR1B7 and AKR1C3 obtained from the respective PGDS and PGFS assays
at pH 7.0 Data are presented as the mean ± SD from four to six independent experiments.
Trang 4151 nmolÆmin)1Æ(mg protein))1, respectively, for PGDS
activity in the absence of NADPH or NADP+ at
pH 8.5 However, AKR1B3 showed a Km value of
33 lm and Vmax value of 240 nmolÆmin)1Æ(mg
pro-tein))1 for PGFS activity at pH 5.0, and 18 lm and
57 nmolÆmin)1Æ(mg protein))1, respectively, for PGDS
activity at pH 8.5 (Fig S1B) Similar affinities for the
substrate PGH2 and Vmax values of PGFS and PGDS
activities of AKR1B1 and AKR1B3 suggest that the
substrate is bound in a similar fashion
Mutagenesis analyses of the effect of the AKR
tetrad in AKR1B1 on PGDS and PGFS activities
X-Ray crystallographic and biochemical analyses of
AKR1B1 revealed that this protein contains a catalytic
tetrad composed of Asp43, Tyr48, Lys77 and His110,
which is highly conserved among members of the
AKR family and constructs the common active site
with a hydrophobic core in this family [3–6] To
iden-tify the catalytic residues involved in the PGDS and
PGFS activity catalyzed by AKR1B1, we introduced
site-directed mutagenesis into the tetrad, generating the
D43N, D43E, Y48F, K77L, K77R, H110F and
H110A mutants, and assessed their PGDS and PGFS
activities with 5 lm [1-14C]PGH2 at the optimum
pH 8.5 for PGDS activity and pH 5.5 for PGFS
activ-ity in the absence and presence of 0.5 mm NADPH,
respectively The typical autoradiograms of TLC used
for PGDS and PGFS assays are shown in Fig 3A,B,
respectively Under these conditions, the Y48F mutant
changed slightly both PGDS and PGFS activities from
wild-type AKR1B1 (138% and 69% of wild-type
AKR1B1, respectively), although this mutant decreased
the p-nitrobenzaldehyde reductase activity to 0.2%
(Fig 3C), indicating that the catalytic Y48 residue is
essential for AKR activity but not necessary for either
PGDS or PGFS activity However, all other mutants
of the tetrad, including H110, showed some trace
activity on both PGDS and PGFS activities (Fig 3C),
suggesting that the triad of Asp43, Lys77 and His110
residues is essential for these activities
When site-specific mutagenesis was introduced to
Y48 and H110 of AKR1B3 (Fig S2C), the Y48F
mutant changed slightly both PGDS and PGFS
activi-ties (133% and 286% of wild-type AKR1B3,
respec-tively), and the H110F mutant decreased the PGDS
and PGFS activities to 37% and 1%, respectively The
double mutant Y48F⁄ H110F completely lost PGDS
activity and showed a weak PGFS activity (2.9%)
These results suggest that both Tyr48 and His110
resi-dues are essential for PGDS activity in the case of
AKR1B3 (Fig S2A–C)
The p-nitrobenzaldehyde reductase activity of AKR1B1 [291 nmolÆmin)1Æ(mg protein))1 at pH 7.0, Fig 3C] was decreased remarkably to less than 1% in the Y48F, K77L and H110F mutants, to 4% in the D43N mutant and to 12% in the H110A mutant The AKR activity of the D43N and K77L mutants was partly restored in the charge-recovered (D43E and K77R) mutants to 41% and 20%, respectively (Fig 3C) However, the p-nitrobenzaldehyde reductase activity of AKR1B3 [542 nmolÆmin)1Æ(mg protein))1at
pH 7.0] was also decreased to less than 1% in the Y48F and H110F mutants (Fig S2C) These results are consistent with previous reports that the p-nitro-benzaldehyde reductase activity of AKR1B1 is
Fig 3 Mutagenesis analyses of the catalytic tetrad of AKR1B1 Typical autoradiogram of TLC used for the PGDS assay at optimum
pH 8.5 (A) and the PGFS assay at the optimum pH 5.5 (B) for AKR1B1 (C) Summarized enzyme activities obtained from the respective PGDS and PGFS assays at the optimum pH and the NADPH-dependent p-nitrobenzaldehyde reductase (PNBR) activity
at pH 7.0 by the wild-type and mutants (D) Typical fluorescence quenching curves of wild-type AKR1B1 and its mutants The bind-ing of NADP + to these proteins was determined by measuring the decrease in fluorescence emission at 338 nm (excitation at
282 nm) Data are presented as the mean ± SD from three to six independent experiments.
Trang 5catalyzed by the triad composed of Tyr48, Lys77 and
His110 and assisted by ionic interaction with Asp43
[25,26], and suggest that Tyr48 is also crucial for the
p-nitrobenzaldehyde reductase activity of AKR1B3
(Fig S2C)
Furthermore, all these mutants of AKR1B1 and
AKR1B3 showed fluorescence quenching of intrinsic
Trp residues after incubation with NADP+ in a
con-centration-dependent manner The Kd values of
NADP+ for AKR1B1 and AKR1B3 are summarized
in Tables 1 and S1, respectively, and the typical
fluo-rescence quenching curves of AKR1B1 and its mutants
are shown in Fig 3D The Kd value of the K77L
mutant of AKR1B1 was similar to that of the
wild-type enzyme (0.3 lm) and the values of the D43N,
D43E, Y48F, K77R, H110F and H110A mutants were
12–360 times higher than that of the wild-type
AKR1B1 However, the Kd value of the Y48F⁄ H110F
double mutant of AKR1B3 was similar to that of the
wild-type enzyme (5.7 lm) and the values of the Y48F
and H110F mutants were 12 times higher than that of
the wild-type AKR1B3 These results confirm that
these mutations do not affect significantly the overall
three-dimensional structure of the cofactor-binding site
within the catalytic pocket
Discussion
Catalytic mechanism of PGDS and PGFS
activities of AKR1B1
Mutational analysis of the catalytic tetrad of AKR1B1
and pH titration analysis revealed that the His110
resi-due functioned as a proton acceptor and donor during
the conversion of PGH2 to PGD2 and PGF2a,
respec-tively pH titration analysis of PGDS and PGFS
activ-ities demonstrated that PGD2 formation required a
deprotonated group with a pKa value of 7.9 for
AKR1B1, and PGF2aformation required a protonated
group with a pKb value of 6.2 (Fig 2A) In the light
of the expected acidity, His110 was deduced to act as the proton acceptor and donor for PGH2 to produce PGD2 and PGF2a, respectively, at physiologic pH, because the imidazolium side chain of His has a pKa value in the range 6–7, whereas the value for the hydroxyl group of Tyr is about 10, and those of Asp and Lys are about 3.6 and 10.5, respectively
Molecular docking simulation of PGH2 to the cry-stallographic structure of AKR1B1 (PDB code, 2qxw; resolution, 0.8 A˚) demonstrated that the substrate PGH2 was bound to the substrate-binding cavity in an extended conformation at the top of the (a⁄ b)8-barrel (Fig 4A,B) The docking calculation, including molec-ular dynamics, revealed that the 11-endoperoxide oxy-gen atom of PGH2was accessible to His110 within the AKR tetrad at a distance of 2.9 A˚, and the substrate PGH2 was stabilized by both hydrophobic and hydro-philic interactions with Trp20, Val47, Trp79, Trp111, Phe122, Pro218, Trp219 and Leu300 (Fig 4C) In the presence of NADP+, when H atoms were added to the protein crystal structure of AKR1B1 by the myP-resto⁄ tplgene program [27] and used for the construc-tion of the energy minimizaconstruc-tion model by the cosgene molecular dynamics simulation program, the distance between the H atom of the OH group (O34) of NADP+ and the carboxyl O atom of Asp43 was 2.61 A˚, within a hydrogen-bonding distance
These results suggest that the His110 residue is the catalytic residue of PGDS and PGFS activity The role
of Lys77 could be to deprotonate the protonated His110, but it might just form a stable hydrogen bond,
or a hydrogen bond network around the active site, to assist acid–base catalysis The Asp43 residue is also important for the hydrogen bond network Further-more, the observation that Km for NADP+ is signifi-cantly altered in these mutants also suggests that Lys77 and Asp43 may have roles in NADPH binding
as well as catalysis The hypothetical catalytic mecha-nisms of PGDS and PGFS activities of AKR1B1 are shown schematically in Fig 5 In the absence of NADPH, the concerted reaction of Asp43, Lys77 and His110 increases the basicity of His110 and extracts the proton C11 of PGH2 Another proton is provided
to the O9 atom of PGH2 from an unidentified proton donor (EnzA-H) to produce PGD2 However, in the presence of NADPH, the hydride ion is transferred from NADPH to the O9 atom of the peroxide oxygen
of PGH2, and a proton is provided from His110 to O11 to produce PGF2a In the presence of NADP+, Asp43 forms a hydrogen bond with NADP+ and dis-rupts the catalytic triad, which is essential for the pro-duction of PGD2 However, the function of Tyr48 is not clear at present
Table 1 NADP + -binding affinities of wild-type (WT) and mutants of
AKR1B1.
K d (l M )
Trang 6Comparison of AKR1B1- and AKR1B3-catalyzed
PGDS and PGFS activities with other
AKR-mediated reactions
In the p-nitrobenzaldehyde reductase activity of
AKR1B1 and AKR1B3 in the presence of NADPH,
Tyr48 acts as the proton donor, consistent with
previ-ous reports from the mutational analysis of AKR1B1
[25,26] and various other members of the AKR
super-family [28,29], in which all AKRs have been shown to
retain the same active site, and the conserved Tyr
resi-due in the catalytic tetrad has been identified to play a
crucial role in the catalysis of NADPH-dependent
reduction Alternatively, we propose a mechanism in
the PGDS and PGFS reactions catalyzed by AKR1B1
in which His110 acts as a base in concert with Asp43
and Lys77 to generate PGD2 in the absence of
NADPH or NADP+, and as an acid to generate
PGF2ain the presence of NADPH
However, the H110F mutant of AKR1B3 retained more than 25% of the wild-type PGDS activity, so that we generated a Y48F⁄ H110F double mutant of AKR1B3 This double mutant completely lost PGDS activity and showed only 2.9% of PGFS activity (Fig S2C) These results suggest that both Tyr48 and His110 residues are essential for PGDS activity in the case of AKR1B3, different from AKR1B1 The deter-mination of the X-ray crystallographic structure of AKR1B3 is needed to elucidate the catalytic mecha-nism of PGDS and PGFS activities of AKR1B3
We have reported previously that Trypanosoma bru-cei PGFS (AKR5A2 with 40.1% amino acid sequence identity with human AKR1B1) utilizes His110, but not Tyr48, as the catalytic residue for the reduction of PGH2 to PGF2a in the presence of NADPH There-fore, the catalytic mechanism of PGFS activity of AKR1B1, AKR1B3 and T brucei PGFS may be con-sidered to be identical However, T brucei PGFS did
Fig 4 Overall views of the ternary complex
of human AKR1B1–NADPH–PGH2 The
three-dimensional structure was calculated
by the program sievgene ⁄ myPresto [http://
medals.jp/myPresto/index.html; http://
presto.protein.osaka-u.ac.jp/myPresto4/].
(A) Schematic drawing of the ternary
structure using the program PyMOL version
1.0 [39] showing the TIM barrel structure of
AKR1B1 (PDB code, 2QXW; resolution,
0.8 A ˚ ; green), NADP +
(yellow), the sub-strate, PGH2(orange), Tyr48 (purple), Asp43
(green), Lys77 (cyan) and His110 (magenta).
(B) Stereoview of the active site model with
the NADPH (yellow) and PGH2(orange)
mol-ecules (stick models) bound in the
active-site cleft consisting of Asp43 (green), Tyr48
(purple), Lys77 (cyan) and His110 (magenta).
(C) Schematic representation of the
interac-tions between PGH 2 (orange) and human
AKR1B1 PGH 2 is predicted to be stabilized
by both hydrophobic (blue) and hydrophilic
(red) interactions with Trp20, Val47, Trp79,
Trp111, Phe122, Pro218, Trp219 and
Leu300.
Trang 7not catalyze PGDS activity in the absence of NADPH
or NADP+ (N Nagata & Y Urade, unpublished
results) These results indicate that PGDS activity is
selective to AKR1B1 and AKR1B3, but not AKR1B7,
AKR1C3 and AKR5A2, and suggest that the tertiary
structure of the catalytic pocket, especially the PGH2
-binding site, of AKR1B1 and AKR1B3 is very similar
and different from that of other members of the AKR
family
Human AKR1B1 has been reported recently to
func-tion as PGFS in the endometrium and is a potential
target for the treatment of menstrual disorders [15],
and mouse AKR1B3 has been reported to be involved
in the suppression of adipogenesis through FP
recep-tors [16] Further characterization of the in vivo
func-tion of AKR1B1 in the endometrium and AKR1B3 in
adipocytes as PGFS is essential to understand the
development of menstrual disorders and metabolic
dis-orders, such as diabetes and obesity, respectively
How-ever, the catalytic mechanisms of PGFS catalyzed by
several isozymes of mammalian AKRs are not clearly
understood, because the X-ray crystallographic
struc-tures of AKR1B3 and AKR1B7 have not yet been
determined Our findings are useful for the design of
inhibitors selective to AKR1B1, which can be employed
for the evaluation of its contribution to the biosynthesis
of PGF2ain various systems
Materials and methods
Expression and purification of recombinant AKR enzymes
Open reading frames of the wild-type enzymes of AKR1B1, AKR1B3, AKR1B7 and AKR1C3, and their mutants, were inserted between NdeI and BamH1⁄ EcoRI sites of the expression vector pET-28a, as described previously [30,31], and used for the transformation of E coli BL21DE3 (Invi-trogen, Carlsbad, CA, USA) The outside primers used for PCR amplifications of the inserts were as follows: 5¢-1B1 NdeI primer (5¢-CGGCAGCCATATGGCAAGCCGTC-3¢) and 3¢-1B1 EcoRI primer (5¢-CGGAATTCGGGCTTCAA AACTCTTCATGG-3¢); 5¢-1B3 NdeI primer (5¢-CGGCA GCCATATGGCCAGCCATC-3¢) and 3¢-1B3 EcoRI primer (5¢-CACGAATTCCAGAGAGACACAGGACACT TGC-3¢); 5¢-1B7 NdeI primer (5¢-CGGCAGCCATATGGC CACCTTCGT-3¢) and 3¢-1B7 BamHI primer (5¢-CGGG ATCCCGTCAGTATTCCTCGTGG-3¢); and 5¢-1C3 NdeI primer (5¢-GGAATTCCATATGGATTCCAAACACCAG TG-3¢) and 3¢-1C3 EcoRI primer (5¢-CGGAATTCTTAA
Fig 5 Schematic drawings of the PGDS (top) and PGFS (bottom) activities in the absence and presence of NADPH, respectively (see Discussion for detailed description).
Trang 8TATTCATCTGAATATG-3¢) Site-directed mutagenesis
was performed using a QuikChange site-directed
muta-genesis kit (Agilent Technologies, Santa Clara, CA, USA)
The D43N-, D43E-, Y48F-, K77L-, K77R-, H110A- and
H110F-substituted recombinant enzymes for AKR1B1 and
the Y48F- and H110F-substituted recombinant enzymes for
AKR1B3 were obtained using the following respective
oligonucleotide primer pairs: AKR1B1 D43N forward (F)
(5¢-GTACCGCCACATCAACTGTGCCCATGTG-3¢) and
reverse (R) (5¢-CACATGGGCACAGTTGATGTGGCGG
TACC-3¢); AKR1B1 D43E (F) (5¢-GGGTACCGCCACA
TCGAATGTGCCCATGTG-3¢) and (R) (5¢-CACATGGG
CACATTCGATGTGGCGGTACCC-3¢); AKR1B1 Y48F (F)
(5¢-CTGTGCCCATGTGTTCCAGAATGAGAATG-3¢) and
(R) (5¢-CATTCTCATTCTGGAACACATGGGCACAG-3¢);
AKR1B1 K77L (F) (5¢-CTTCATCGTCAGCCTGCTGTG
GTGCACG-3¢) and (R) (5¢-CGTGCACCACAGCAGGCT
GACGATGAAG-3¢); AKR1B1 K77R (F) (5¢-CTCTTCA
TCGTCAGCAGGCTGTGGTGCACG-3¢) and (R) (5¢-CG
TGCACCACAGCCTGCTGACGATGAAGAG-3¢); AKR1
B1 H110F (F) (5¢-CCTCTACCTTATTTTCTGGCCGACT
GGC-3¢) and (R) (5¢-GCCAGTCGGCCAGAAAATAAG
GTAGAGG-3¢); AKR1B1 H110A (F) (5¢-CCTCTACCTT
ATTGCCTGGCCGACTGGC-3¢) and (R)
(5¢-GCCAGTC-GGCCAGGCAATAAGGTAGAGG-3¢); AKR1B3 Y48F (F)
(5¢-GACTGCGCCCAGGTGTTCCAGAATGAGAAG-3¢)
and (R) (5¢-CTTCTCATTCTGGAACACCTGGGCGCAG
TC-3¢); AKR1B3 H110F (F) (5¢-GATCTCTACCTTATT
TTCTGGCCAACGGGG-3¢) and (R) (5¢-CCCCGTTGGC
CAGAAAATAAGGTAGAGATC-3¢) (the italic codons
indicate the sites of mutations) Transformed cells were
pre-cultured overnight at 30C Induction was started by the
addition of 1 mm isopropyl thio-b-d-galactoside (final
con-centration, 1 mm) when the absorbance (A) at 600 nm of
the culture had reached 0.5–0.6, and further cultivation was
carried out for 6 h at 30C The recombinant protein was
purified by chromatography with nickel nitrilotriacetate
His-Bind resin (Merck, Darmstadt, Germany) according to
the manufacturer’s protocol, followed by digestion with
thrombin to remove the 6· His tag The recombinant
protein was further purified by gel filtration
chromatogra-phy with Hiload 16⁄ 60 Superdex 75 pg (GE Healthcare,
Amersham, Buckinghamshire, UK) in Dulbecco’s
phos-phate-buffered saline Protein purity was assessed by
SDS⁄ PAGE on 10–20% gradient gels after staining with
Coomassie Brilliant Blue Protein concentrations were
mea-sured using a BCA Protein Assay Kit (Pierce
Biotechnol-ogy, Rockford, IL, USA)
Enzyme activity assays
The PGFS and PGDS activities of AKR proteins were
determined as described previously [32] In brief, the purified
recombinant enzymes were incubated at 37C for 2 min
with 5 lm 1-[14C]PGH2 as a substrate in the presence or
absence of 0.5 mm NADPH in 50 mm sodium phosphate,
pH 7.0 The reaction was terminated by the addition of
300 lL of diethyl ether–methanol–2 m citric acid (30 : 4 : 1,
v⁄ v ⁄ v) The reaction products recovered into the organic phase were separated by TLC The conversion rate from 14
C-labeled substrate to14C-labeled product was calculated using an imaging plate system (Fuji Film, Tokyo, Japan) The kinetic constants were determined from Lineweaver– Burk plots prepared with sigmaplot software (version 10.0 for Windows; Systat Software, Inc., San Jose, CA, USA) For pH–rate profiles, Kmvalues were calculated from ini-tial velocity studies over a wide range of pH values using a triple buffer system containing 50 mm sodium phosphate,
50 mm sodium pyrophosphate and 50 mm 3-[(1,1-dimethyl-2-hydroxyethl)amino-2-hydroxypropanesufonic acid In analyzing these data, the pKa and pKb values were esti-mated using the fitting equation
y¼ ½C=ð1 þ 10ðpKapHÞþ10ðpHpKbÞ ð1Þ
prepared with sigmaplot software C is the pH-indepen-dent value of V The p-nitrobenzaldehyde reductase activity
of AKR1B1 was measured with 0.2 mm NADPH and
1 mm p-nitrobenzaldehyde in 100 mm sodium phosphate (pH 7.0) The reaction was initiated by the addition of the substrate, and the decrease in the absorbance at 340 nm was monitored at 25C [9]
Fluorescence quenching assay
The binding of NADP+to wild-type and mutant proteins
of AKR1B1 was determined by performing a fluorescence quenching assay, in which various concentrations of coen-zyme were incubated with AKR1B1 proteins in 300 lL of
50 mm sodium phosphate (pH 7.0) at 25C for 2 min The intrinsic Trp fluorescence was measured using an FP-6200 spectrofluorometer (JASCO, Tokyo, Japan) operated at an excitation wavelength of 282 nm and an emission wave-length of 338 nm [33] The Kdvalues for coenzyme binding
to AKR1B1 proteins were calculated from the difference in fluorescence signal observed in the presence and absence
of coenzyme, as reported previously [26], with sigmaplot software
Molecular docking simulation
The docking study was performed by sievgene⁄ myPresto (http://medals.jp/myPresto/index.html; http://presto.pro-tein.osaka-u.ac.jp/myPresto4/) [27] The prediction accura-cies of the sievgene program have already been reported to
be 19.2%, 50.78% and 60.0% with rmsd values of less than
1 A˚, 2 A˚ and 3 A˚, respectively, in a total of 130 complexes Among the top 10 docking models, the probabilities increase to 28.5%, 63.1% and 76.9% with rmsd values of less than 1 A˚, 2 A˚ and 3 A˚, respectively [27] The initial
Trang 9three-dimensional coordinates of the small compounds were
generated by the Chem3D program (cambridge Software,
Cambridge, MA, USA) manually We used the general
AMBER force field [34], and the molecular topology files
were generated by tplgeneL⁄ myPresto The energy
optimi-zation of the coordinates of small compounds was
per-formed using cosgene⁄ myPresto [35] The atomic charges
were calculated by the Gasteiger method of Hgene⁄
myPres-to [36,37] The amyPres-tomic charges of the proteins were the
same as the atomic charges of AMBER parm99 [38] For
flexible docking, the sievgene program generated up to 1000
conformers for each compound We predicted that the C4
atom of nicotinamide reacts with the O atom of PGH2and
that these two atoms should be close to each other Among
the top 10 docking models, two models similar to each
other showed C4–O distances of 2.0 and 2.1 A˚, consistent
with the experimental results, whereas the other eight
mod-els showed C4–O distances of more than 6.5 A˚ The
com-plex structure depicted in Fig 4 was given by the energy
minimization calculation based on the model with a C4–O
distance of 2.0 A˚ The position of the ring structure of PG
should be 70–80% accurate based on the prediction
accu-racy of ‘sievgene’ Although it is difficult to predict the
position of side chains of PG, the side chains are not
important for the discussion of the reaction mechanism
Acknowledgements
This work was supported in part by the Japan
Aero-space Exploration Agency (JAXA), the Program
of Basic and Applied Researches for Innovations in
Bio-oriented Industry of Japan, Takeda Science
Foun-dation, and Osaka City (to Y.U.) and Grant-in-Aid for
Scientific Research (No 22550152) from the Ministry
of Education, Culture, Sports, Science and Technology
of Japan (to T.I.) We thank Dr Michele Manin (CNRS
UMR6247-GReD, France) for kindly providing the
AKR1B1 expression vector; Drs Kenji Mizuguchi and
Sukanta Mondal (National Institute of Biomedical
Innovation, Ibaraki, Japan) for homology modeling;
Dr Toshiyoshi Yamamoto (Department of Molecular
Biology and Medicine, Research Center for Advanced
Science and Technology, University of Tokyo, Japan)
for kinetic analysis; Dr Zakayi Kabututu, Nobuko
Uodome and Toshiharu Tsurumura (Osaka Bioscience
Institute, Japan) for assistance during the early stage of
this research; and Megumi Yamaguchi, Naoko
Takah-ashi and Taeko Nishimoto (Osaka Bioscience Institute,
Japan) for secretarial assistance
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