Role of glutamine 148 of human 15-hydroxyprostaglandin dehydrogenase in catalytic oxidation of prostaglandin E2

Article abstract of DOI:10.1016/j.bmc.2006.06.030

NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins. This enzyme oxidizes the C-15 hydroxyl group of prostaglandins and lipoxins to produce 15-keto metabolites which exhibit greatly reduced biological activities. A three-dimensional (3D) structure of 15-PGDH based on the crystal structures of the levodione reductase and tropinone reductase-II was generated and used for docking study with NAD⁺ coenzyme and PGE₂ substrate. Three well-conserved residues among SDR family which correspond to Ser-138, Tyr-151, and Lys-155 of 15-PGDH have been shown to participate in the catalytic reaction. Based on the molecular interactions observed from 3D structure of 15-PGDH, we further propose that Gln-148 in 15-PGDH is important in properly positioning the 15-hydroxyl group of PGE₂ by hydrogen bonding with the side-chain oxygen atom of Gln-148. This residue is found to be less conserved and replaceable by glutamyl, histidinyl, and asparaginyl residues in SDR family. Accordingly, site-directed mutagenesis of Gln-148 of 15-PGDH to alanine, glutamic acid, histidine, and asparagine (Q148A, Q148E, Q148H, and Q148N) was carried out. The activity of mutant Q148A was not detectable, whereas those of mutants Q148E, Q148H, and Q148N were comparable to or higher than the wild type. This indicates that the side-chain oxygen or nitrogen atom at position 148 of 15-PGDH plays an important role in anchoring C-15 hydroxyl group of PGE₂ through hydrogen bonding for catalytic reaction.

Full text of DOI:10.1016/j.bmc.2006.06.030

Bioorganic & Medicinal Chemistry 14 (2006) 6486–6491
Role of glutamine 148 of human 15-hydroxyprostaglandin
dehydrogenase in catalytic oxidation of prostaglandin E2
Hoon Cho,a,b Lingyu Huang,b, Adel Hamza,b Daquan Gao,b
Chang-Guo Zhanb,* and Hsin-Hsiung Taib,*
aPolymer Science Engineering, Chosun University, Gwangju 501-579, Republic of Korea
bDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA
Received 25 March 2006; revised 3 June 2006; accepted 8 June 2006
Available online 7 July 2006
Abstract—NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/re-
ductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins. This enzyme oxidizes the
C-15 hydroxyl group of prostaglandins and lipoxins to produce 15-keto metabolites which exhibit greatly reduced biological activ-
ities. A three-dimensional (3D) structure of 15-PGDH based on the crystal structures of the levodione reductase and tropinone
reductase-II was generated and used for docking study with NAD+ coenzyme and PGE2 substrate. Three well-conserved residues
among SDR family which correspond to Ser-138, Tyr-151, and Lys-155 of 15-PGDH have been shown to participate in the catalytic
reaction. Based on the molecular interactions observed from 3D structure of 15-PGDH, we further propose that Gln-148 in
15-PGDH is important in properly positioning the 15-hydroxyl group of PGE2 by hydrogen bonding with the side-chain oxygen
atom of Gln-148. This residue is found to be less conserved and replaceable by glutamyl, histidinyl, and asparaginyl residues in
SDR family. Accordingly, site-directed mutagenesis of Gln-148 of 15-PGDH to alanine, glutamic acid, histidine, and asparagine
(Q148A, Q148E, Q148H, and Q148N) was carried out. The activity of mutant Q148A was not detectable, whereas those of mutants
Q148E, Q148H, and Q148N were comparable to or higher than the wild type. This indicates that the side-chain oxygen or nitrogen
atom at position 148 of 15-PGDH plays an important role in anchoring C-15 hydroxyl group of PGE2 through hydrogen bonding
for catalytic reaction.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
ic, while Type II is NADP+ preferred. Type I is more
prostaglandin and lipoxin specific and exhibits a low
Km for prostaglandins and lipoxins, whereas Type II
has a much broader substrate specificity and shows a
high Km for prostaglandins.2 In fact, Type II was later
found to be identical with carbonyl reductase.3 There-
fore, Type I has been considered the key enzyme respon-
sible for the biological inactivation of prostaglandins
and lipoxins. Studies on prostaglandin and lipoxin
catabolism have so far been focused on the Type I
enzyme.
Prostaglandins and lipoxins are two families of biologi-
cally potent fatty acids derived from arachidonic acid
through cyclooxygenase and lipoxygenase pathways,
respectively. Prostaglandins and lipoxins are rapidly
metabolized by the initial oxidation of the 15(S)-hydroxyl
group
catalyzed
by
15-hydroxyprostaglandin
dehydrogenase (15-PGDH).1 Two different types of
15-PGDH have been recognized. Type I is NAD+ specif-
Abbreviations: 15-PGDH, NAD+-dependent 15-hydroxyprostaglandin
dehydrogenase; SDR, short-chain dehydrogenase/reductase; DTT, di-
thiothreitol; PVDF, polyvinylidene; GST, glutathione S-transferase;
SCR, structural conserved regions; VR, variable regions.
Type I 15-PGDH (hereafter referred to as 15-PGDH)
is active as a homodimer with a subunit Mw of
29 kDa.4 Sequence alignments indicate that it belongs
to the short-chain dehydrogenase/reductase (SDR)
family. A consensus sequence of Tyr-X-X-X-Lys as
well as a nearby generally conserved upstream serine
residue in the middle part of the enzymes character-
izes this protein family.5 Furthermore, a glycine-rich
motif Gly-X-X-X-Gly-X-Gly typical of a Rossmann
Keywords: Prostaglandins; 15-Hydroxyprostaglandin dehydrogenase;
Molecular modeling; Site-directed mutagenesis.
*
Corresponding authors. Tel.: +859 257 1837; fax: +859 257 7585;
e-mail: htai1@uky.edu
Present address: Human Nutrition and Health Program, Kentucky
State University, Frankfort, KY 40601, USA.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.030

H. Cho et al. / Bioorg. Med. Chem. 14 (2006) 6486–6491
6487
fold is found near the N-terminus and is thought to
be part of the putative coenzyme binding site.6 Three
well-conserved residues among SDR family (the triad
Ser-138, Tyr-151, and Lys-155 in 15-PGDH) are
known to be involved in the catalytic reaction, based
on mutagenesis7–13 and X-ray analysis.14–21 We also
have identified Tyr-151, Lys-155, and Ser-138 in 15-
PGDH as being a catalytically important triad by
site-directed mutagenesis.22,23 Tyr-151 is the most
conserved residue in SDR and probably acts as a
general base in the catalytic reaction. Lys-155 has a
dual role: fixation of the nicotinamide ring of the
coenzyme and lowering the pKa value of the phenolic
group of Tyr-151. Finally, Ser-138 is also conserved
in the SDR family and assists the proper positioning
of the hydroxyl or carbonyl group of the substrate.
We have also identified Thr-11, Ile-74, Asn-91,
Cys-182, Val-186, and Thr-188 in 15-PGDH as being
involved in interacting with NAD+ by site-directed
mutagenesis.24–27
structural feature of the proteins or involvement in the
catalytic mechanism. Several conserved residues in every
single SDR enzyme presently known such as residues
corresponding to Ser-138,8 Tyr-151,7 Lys-155,7
Cys-182,12 and Thr-18825 of human placental 15-PGDH
were found to be essential for catalytic activity. Recent-
ly, we also found that Gln-15, Asp-36, and Trp-37 of
human placental 15-PGDH appeared to be involved in
coenzyme specificity.30
Although the crystal structure of 15-PGDH is yet to be
determined, the crystal structures of the binary and ter-
tiary complexes of other members of the SDR family,
such as levodione reductase from C. aquaticum and
tropinone reductase-II from D. stramonium have been
elucidated. A 3D structure of 15-PGDH based on the
crystal structures of the levodione reductase and tropi-
none reductase-II was generated and used for docking
study with NAD+ coenzyme and PGE2 substrate.
Sequence alignment of levodione reductase, tropinone
reductase-II, and 15-PGDH as well as other SDR
enzymes is shown in Figure 1. Ser-152, Gln-162,
Tyr-165, and Lys-169 of levodione reductase and Ser-
146, Gln-156, Tyr-159, and Lys-163 of tropinone reduc-
tase-II which correspond to Ser-138, Gln-148, Tyr-151,
and Lys-155 of 15-PGDH were shown to make interac-
tions with cofactor and substrate. By examining the
high-resolution crystal structures of the apo form,
binary and ternary complexes, and biochemical data
gathered for many SDR family members, we have pro-
posed a reaction mechanism for 15-PGDH.7–21 At pres-
ent, the ‘Ser-Tyr-Lys catalytic triad’ is considered to be
important in SDR catalysis, whereby the side-chain oxy-
gen of the tyrosine residue functions as an acid–base cat-
alyst for proton transfer. These conserved residues are in
fact located around the hydroxyl group of PGE2 in the
ternary complex of 15-PGDH, and the side-chain oxy-
gen of Tyr-151 residue points toward the face of the
cofactor nicotinamide ring. We have identified Ser-138,
Tyr-151, and Lys-155 in 15-PGDH as being a catalyti-
cally important triad by site-directed mutagenesis7,8
and homology modeling.31 By homology modeling,
docking, molecular dynamics simulation, and site-di-
rected mutagenesis of 15-PGDH, we also can propose
that the Gln-148 in 15-PGDH is important in the proper
positioning of the C-15 hydroxyl group of PGE2 sub-
strate during catalytic reaction.
Enzymes in the SDR family are either NAD+ or
NADP+ specific or utilize both coenzymes. Either
nucleotide stereospecifically transfers a hydride from
or to the carbon atom of the substrates in position
4 of the nicotinamide ring. Although the three-di-
mensional structure of 15-PGDH has yet to be
determined, crystal structures of the binary and
tertiary complexes of other members of the SDR
family, such as levodione reductase from Corynebac-
terium aquaticum28 and tropinone reductase-II from
Datura stramonium,29 have been elucidated. A 3D
structure of 15-PGDH based on the crystal struc-
tures of the levodione reductase and tropinone
reductase-II was generated and used for docking
study with the NAD+ coenzyme. A ternary struc-
ture-based sequence alignment with 10 members of
the SDR family of known three-dimensional struc-
tures has suggested a model which relates the ob-
served low degree of sequence identity to quite
similar folding patterns and nearly identical distribu-
tions of residues involved in catalysis. The reliability
of this model is supported by our publications of
studies on 15-PGDH active sites using site-directed
mutagenesis. From the 3D structural analysis of
15-PGDH-NAD+-PGE2 complex and the sequence
analysis of the SDR family, we propose that Gln-
148 participates in the catalytic oxidation of PGE2
in 15-PGDH. Site-directed mutagenesis study of this
residue supports this contention as revealed in the
current report.
A proposed 3D model of a tertiary complex of
15-PGDH-NAD+-PGE2 is shown in Figure 2. The mod-
el shows that the 15-hydroxyl group of the PGE2 is an-
chored by hydrogen bonding with the hydroxyl group of
Ser-138 and the side-chain oxygen atom of Gln-148.
Among the SDR enzymes listed in Figure 1, residue cor-
responding to Ser-138 is basically conserved, whereas
that corresponding to Gln-148 can be glutamine as well
as glutamic acid, histidine, and asparagine. Based on
this information, site-directed mutagenesis of Gln-148
of 15-PGDH to alanine, glutamic acid, histidine, and
asparagine (Q148A, Q148E, Q148H, and Q148N) was
carried out (Table 1). The activity of mutant Q148A
was not detectable, whereas those of mutants Q148E,
Q148H, and Q148N were comparable to or higher than
2. Results and discussion
15-PGDH is a member of the SDR family of about 60
enzymes.5 Even though the overall homology among
these enzymes is low, there are a number of amino acid
residues which are highly conserved. Conservation of
these residues would suggest that they serve a common
role in all of the proteins in which they are present.
The roles of these residues might include binding of
the nucleotide cofactor, conservation of an important

6488
H. Cho et al. / Bioorg. Med. Chem. 14 (2006) 6486–6491
138 148 151
155
15-PGDH
130 G G I I I N M S S L A G L M – P V A Q Q P V Y C A S K H G I V G F T R 163
LevoR
TR-II
144 S G M V V N T A S V G G I R – G I G N Q S G Y A A A K H G V V G L T T 177
138 R G N V V F I S S V S G A L – A V P Y E A V Y G A T K G A M D Q L T R 171
17β-HSD-1
NPG
3-O(K)AR
FabG
134 S G R V L V T G S V G G L M – G L P F N D V Y C A S K F A L E G L C E 167
132 W G R I I N I S S V N G V K – G Q A G Q T N Y S A A K A G V I G F T K 165
128 F G R I I N I G S V V G S T – G N P G Q T N Y C A A K A G V V G F S K 161
128 F G R I I T I G S V V G S M – G N P G Q T N Y C A A K A G L I G F S K 161
132 T G D I I V T C S I A G H F – P T Y W E P V Y S G S K W A I T S F V Q 165
124 R G H V V N I S S I A G A Y – N F G G N S T Y H A T K A A I H M L S R 157
131 N G L V I N V S S G L S N L – A D G A H P I Y N L T K A G V H F Y S D 164
132 F G R I V N V A S L A G A V P G A A G H T L Y A A T K G F L V K F S Q 166
144 S G M V V N T A S V G G I R – G I G N Q S G Y A A A K H G V V G L T R 177
135 E G R I I S I S S I I G Q A – G G F G Q T N Y S A A K A G M L G F T K 168
135 G G R V I N I S S I I G Q A – G G F G Q T N Y S A A K A G M L G F T K 168
137 G G S I V N I S S V S G F R – P Q P N N I A Y V A A K H G V V G M T K 170
AGR-L
POxidoR
LOxidoR
OxidoR
LDR
ACoR
3-KCoAR
Glu-1-D
Figure 1. Comparison of sequences of the SDR family. Following enzyme sequences are co-listed with that of human 15-PGDH in the figure.
Levodione reductase from Corynebacterium aquaticum (LevoR); tropinone reductase-II from Datura stramonium (TR-II); human 17b-hydroxysteroid
dehydrogenase type 1 (17b-HSD-1); Azospirillum brasilense nodulation protein G (NPG); Haemophilus influenzae 3-oxoacyl-[acyl-carrier-protein]
reductase (3-ketoacyl-acyl carrier protein reductase) [3-O(K)AR]; Pasteurella multocida FabG (FabG); Agrobacterium tumefaciens str.
AGR_L_1076p (AGR-L); Ralstonia solanacearum Probable oxidoreductase (POxidoR); Lactococcus lactis subsp. lactis oxidoreductase (LOxidoR);
Caulobacter crescentus oxidoreductase (OxidoR); Leifsonia aquatica levodione reductase ((6R)-2,2,6-trimethyl-1,4-cyclohexanedione reductase)
(LDR); Bacillus cereus acetoacetyl-CoA reductase (ACoR); Bacillus megaterium 3-ketoacyl-CoA reductase PhaB (3-KCoAR); Oceanobacillus
iheyensis glucose 1-dehydrogenase (Glu-1-D).
the wild type. This indicates that the side-chain oxygen
or nitrogen atom at position 148 plays an important role
in anchoring 15-hydroxyl group of PGE2 through the
hydrogen bonding. Figure 2 indicates that the Ser-138
and Gln-148 interact with the 15-hydroxyl group of
PGE2 substrate. As seen in Figure 3, molecular dynam-
ics (MD) simulations on mutants Q148A, Q148E,
Q148H, and Q148N were carried out to study the inter-
actions of PGE2 with the side-chains of mutants. The
results showed that the side-chain of the mutant
Q148A (Fig. 3A) cannot form hydrogen bonding with
the 15-hydroxyl group of PGE2 resulting in a wobbling
motion of the hydroxyl group. However, Figures 3(B, C,
and D) indicates that the C-15 hydroxyl group of PGE2
is stabilized by the side-chain oxygen atom of mutants
Q148E and Q148N or by the side-chain nitrogen atom
Figure 2. Constructed 3D structural model of 15-PGDH-NAD+-PGE2
complex. The model was generated by homology molecular modeling
of the mutant Q148H.
based on the crystallographic structures of levodione reductase and
Mutants Q148E, Q148H, and Q148N had higher activi-
tropinone reductase-II. The model indicates the interactions of
ty than the wild type. Especially, the activities of mu-
tants Q148H and Q148N were increased nearly 6-fold
(Table 2). However, kcat/Km ratios of mutants Q148H
hydroxyl group of PGE2 with residues Ser-138 and Gln-148 of the
wild type 15-PGDH.
and Q148E were decreased 3-fold for PGE2 and
3- and 41-fold for NAD+, respectively, because of signif-
icant increase in Km for NAD+ with both mutants.
These results indicate that Gln-148 of the 15-PGDH is
involved in the interaction with both PGE2 and NAD+
and contributes to the full catalytic activity of
15-PGDH.
Table 1. 15-PGDH activity in E. coli expressing GST fusion enzymes
of wild type and its mutants
Transformant
Activity NADH
(lM/min/lg protein)
pGEX-2T-GST-PGDH
2.8
ND
7.5
pGEX-2T-GST-PGDH Q148A
pGEX-2T-GST-PGDH Q148E
pGEX-2T-GST-PGDH Q148H
pGEX-2T-GST-PGDH Q148N
16.0
17.8
In summary, the side-chain oxygen or nitrogen atom of
the amino acid at position 148 may play an important
role in catalytic oxidation of PGE2 as glutamic acid, his-
tidine, and asparagine mutants exhibited higher activity
and alanine mutant showed no activity at all. C-15
hydroxyl group is placed in between oxygen atoms of
the side-chains of Ser-138 and Gln-148. A proton can
then be easily transferred from the alcohol group of
The enzyme was assayed fluorometrically as described in Section 3.
The concentration of PGE2 and NAD+ used was 17.4–99.4 lM and
60–4000 lM, respectively. 15-PGDH and its mutants were expressed as
a GST fusion enzyme using pGEX-2T vector as described in Section 3.
pGEX-2T-GST-PGDH: wild type 15-PGDH. Notation of mutants is
indicated in a similar manner except adding the site of mutation and
the amino acid being mutated to. ND: No detectable activity.

H. Cho et al. / Bioorg. Med. Chem. 14 (2006) 6486–6491
6489
Figure 3. Constructed 3D structural model of 15-PGDH-NAD+-PGE2 complex. The model was generated by homology molecular modeling based
on the crystallographic structures of levodione reductase and tropinone reductase-II. The model indicates the interactions of hydroxyl group of PGE2
with residue Ala-148 of the mutant Q148A (A), the residue Glu-148 of the mutant Q148E (B), the residue His-148 of the mutant Q148H (C), and the
residue Asn-148 of the mutant Q148N (D).
Table 2. Km and kcat/Km values for 15-PGDH mutants
PGE2
kcat(sꢀ1
50
NAD+
kcat (sꢀ1
47
Km (lM)
)
kcat/Km (Mꢀ1 sꢀ1
)
Km (lM)
38
)
kcat/Km (Mꢀ1 sꢀ1
)
Wild type
Q148H
Q148N
Q148E
3.4
58.4
24.5
25.8
0
8
3
4
6
1.5 · 107
0.6 · 107
1.2 · 107
0.5 · 107
4
4
1.24 · 106
0.78 · 106
1.25 · 106
0.03 · 106
329 57
297 32
125 26
342 39
240 20
3273 331
266 26
299 51
112 30
The Km value of the purified enzymes was determined by Lineweaver-Burk plots. Data processing and curve fitting were assisted by using
GRAPHPAD software. The Km value for PGE2 was determined using NAD+ (60–4000 lM) as a coenzyme. The Km value for NAD+ was determined
using PGE2 (17.4–99.4 lM) as a substrate. The enzyme activity was assayed fluorometrically as described in Section 3.
PGE2 to the unprotonated form of residue Tyr-151. This
process facilitates a hydride transfer from the C-15 posi-
tion of the PGE2 to the C-4 position of the nicotinamide
ring of NAD+ (Fig. 4). Ser-138 and Gln-148 either
orients the PGE2 properly or stabilizes the transition
intermediate during the oxidation process or both.
The QIAprep Spin Plasmid Miniprep Kit was from
QIAGEN. ECL+ Plus Western blotting detection system
RPN 2132 was from Amersham Life Science. Oligonu-
cleotide primers were synthesized by the Macromolecu-
lar Structure and Analysis Facility of the University of
Kentucky.
3.2. Molecular modeling of the 15-PGDH
3. Experimental
3.1. Materials
The amino acid sequences of the human 15-hydroxypro-
staglandin dehydrogenase (15-PGDH) [AC: 31542939]
were derived from cDNA sequence as described previ-
ously.4 Based on the crystal structure of the levodione
reductase from C. aquaticum28 and Tropinone reduc-
tase-II from D. stramonium,29 homology-based enzyme
model was built using the program Insight II (Accelrys,
San Diego, CA). In order to find structurally conserved
regions, sequences of the reference proteins were manu-
ally aligned based on its secondary structure. The
sequence of the corresponding human 15-PGDH pro-
tein was then aligned to the reference proteins and the
NAD+, NADH, glutathione (GSH)-Sepharose 4B, sodi-
um dodecylsulfate (SDS) and dithiothreitol (DTT) were
obtained from Sigma. PGE2 was from Cayman Chemi-
cals. Cloned pfu DNA polymerase was from Stratagene.
DpnI endonuclease was from New England BioLabs.
Protein A-HRP was from Transduction Laboratories.
Polyvinylidene fluoride (PVDF) membrane was ob-
tained from the Millipore Corp. GST gene fusion
pGEX-2T expression vector was from Pharmacia Corp.

6490
H. Cho et al. / Bioorg. Med. Chem. 14 (2006) 6486–6491
O
O
COOH
COOH
HO
HO
O
H
H2N
O
GLN148
H
O
OH
O
SER138
O
TYR151
H2N
N+
O
OH
H3N+
LYS155
OH
NAD+
Figure 4. Proposed catalytic mechanism of 15-PGDH.
coordinates were assigned within the structural con-
served regions (SCR) as we did in modeling the wild
type enzyme.31 Loops or variable regions (VR), which
are located between SCRs, were found exclusively at
the surface of the protein, not interacting with the active
site. Coordinates for the VRs were either directly gener-
ated or assigned from known crystal structures. The
conformations of the side chains were retained in con-
served positions and the statistically most likely rotamer
(rotational position of side-chain based on analysis of all
known protein structures containing that amino acid)
was chosen when no conformational information was
available. The final structure was refined by energy min-
imization using the Discover/InsightII with Amber force
field.32 The multiple sequence alignment was performed
using CLUSTALW33 implemented in the BIOEDIT
program.34 All color figures shown are generated using
Weblab viewer (Accelrys, San Diego, CA).
CCC GTT GCA CAG GAA CCG GTT TAT TGT
GCT TCA AAG 30 and 50 CTT TGA AGC ACA
ATA AAC CGG TTC CTG TGC AAC GGG CAT
GAG TCC 30, where the underlined bases indicate the
bases that were changed. Pfu DNA polymerase was used
for PCR. The PCR product was treated with DpnI endo-
nuclease to digest the parental DNA template. The
DNA sequences of the mutants were confirmed by
DNA sequencing.
3.4. Expression and purification of 15-PGDH
Expression and purification of 15-PGDH were carried
out as described previously.26,27 Briefly, the altered
cDNA prepared above was inserted between BamHI
and EcoRI sites of the pGEX-2T expression vector.26
The recombinant plasmid was used to transform E. coli
BL-21 LysS. Cells were grown in 500 mL YT medium
containing 50 lg/mL ampicillin at 37 ꢁC with shaking
(250 rpm) until OD600 reached 0.6. IPTG was added to
a final concentration of 1 mM and cells were allowed
to grow for additional 12 h at 25 ꢁC. Cells were then
harvested by centrifugation at 5000g for 15 min at
4 ꢁC. The cell pellet was resuspended in 20 mL of cold
cell lysis buffer [PBS buffer (pH 7.4) containing 1 mM
EDTA, and 0.1 mM DTT]. The cells were broken by
sonication. The cell lysate was cleared by centrifugation
at 10,000g for 20 min. The extract was slowly loaded
onto the glutathione Sepharose 4B column which was
equilibrated at 4 ꢁC with column buffer [PBS buffer
(pH 7.4) containing 1 mM EDTA, and 0.1 mM DTT].
After washing with column buffer until the OD280
reached less than 0.005, the 15-PGDH was eluted from
the glutathione Sepharose 4B column by incubation at
room temperature for 5 min with the elution buffer
[50 mM Tris–HCl (pH 8.0) containing 10 mM reduced
glutathione, 1 mM EDTA, and 0.1 mM DTT]. The con-
centration of the purified enzyme was determined and
the purity of the enzyme was assessed by SDS–PAGE.
The recombinant fusion enzyme exhibited a Mw of
approximately 55 kDa.
3.3. Site-directed mutagenesis
All site-directed mutagenesis of human placental
15-PGDH cDNA was performed by QuikChange site-
directed mutagenesis method.35 Four pairs of PCR
primers were used to perform PCR. The oligonucleo-
tides used for mutagenesis had the following sequences:
for mutagenesis of glutamine 148 to alanine 148
(Q148A), 50 GGA CTC ATG CCC GTT GCA CAG
GCT CCG GTT TAT TGT GCT TCA AAG 30 and
50 CTT TGA AGC ACA ATA AAC CGG AGC
CTG TGC AAC GGG CAT GAG TCC 30; glutamine
148 to asparagine 148 (Q148N), 50 GGA CTC ATG
CCC GTT GCA CAG AAA CCG GTT TAT TGT
GCT TCA AAG 30 and 50 CTT TGA AGC ACA
ATA AAC CGG TTT CTG TGC AAC GGG CAT
GAG TCC 30; glutamine 148 to histidine 148
(Q148H), 50 GGA CTC ATG CCC GTT GCA CAG
CAC CCG GTT TAT TGT GCT TCA AAG 30 and
50 CTT TGA AGC ACA ATA AAC CGG GTG
CTG TGC AAC GGG CAT GAG TCC 30; glutamine
148 to glutamic acid 148 (Q148E), 50 GGA CTC ATG

H. Cho et al. / Bioorg. Med. Chem. 14 (2006) 6486–6491
6491
13. Swanson, B. A.; Frey, P. A. Biochemistry 1993, 32, 13231–
13236.
3.5. 15-PGDH assay
14. Auerbach, G.; Herrmann, A.; Gutlich, M.; Fischer, M.;
¨
Jacob, U.; Bacher, A.; Huber, R. EMBO J. 1997, 16,
7219–7230.
15. Ghosh, D.; Wawrzak, Z.; Weeks, C. M.; Duax, W. L.;
Erman, M. Structure 1994, 2, 629–640.
16. Ghosh, D.; Erman, M.; Wawrzak, Z.; Duax, W. L.;
Pangborn, W. Structure 1994, 2, 973–980.
17. Hulsmeyer, M.; Hecht, H.-J.; Niefind, K.; Hofer, B.;
¨
Lindsay, D. E.; Timmis, K. H.; Schomburg, D. Protein
Sci. 1998, 7, 1286–1293.
18. Jo¨rnvall, H.; Persson, B.; Krook, M.; Atrian, S.;
The activity of 15-PGDH was assayed at 37 ꢁC fluoro-
metrically by measuring the formation of NADH at
468 nm following excitation at 340 nm using a Shimadzu
RF-5301PC Fluorescence Spectrophotometer.36 For
determining the Km value for prostaglandin E2
(PGE2), the reaction mixture contained: 50 mM Tris–
HCl (pH 7.5), 0.1 mM DTT, 250 lM NAD+, 20 lg of
purified enzyme, and various concentrations of PGE2
in 2 mL total volume. At least five different concentra-
tions of PGE2, ranging from 4 lM to 21 lM, and a fixed
concentration of NAD+ at 250 lM were used to deter-
mine Km value for PGE2. Each concentration was
assayed in triplicate. For determining Km value for
NAD+, PGE2 at a final concentration of 21 lM was
used with various concentrations of NAD+ ranging
from 25 lM to 250 lM. The spectrophotometer was
standardized using a known concentration of NADH
`
Gonzalez-Duarte, R.; Jeffery, J.; Ghosh, D. Biochemistry
1995, 34, 6003–6013.
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determined by measuring the absorbance at 340 nm
37
using 2M = 6.22 · 103 Mꢀ1 cmꢀ1
.
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