Critical Residues for the Specificity of Cofactors and Substrates in Human Estrogenic 17ß-Hydroxysteroid Dehydrogenase 1: Variants Designed from the Three-Dimensional Structure of the Enzyme
Yi-Wei Huang1,
Isabelle Pineau1,
Ho-Jin Chang,
Arezki Azzi,
Véronique Bellemare,
Serge Laberge2 and
Sheng-Xiang Lin
Medical Research Council Group in Molecular Endocrinology,
Oncology, and Molecular Endocrinology Research Center, Laval University
Medical Center, Québec, Québec G1V 4G2, Canada
Address all correspondence and requests for reprints to: Sheng-Xiang Lin, Medical Research Council Group in Molecular Endocrinology, Oncology, and Molecular Endocrinology Research Center, Laval University Medical Center, 2705 Boulevard Laurier, Québec, Québec G1V 4G2, Canada. E-mail: sxlin{at}crchul.ulaval.ca
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ABSTRACT
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Human estrogenic 17ß-hydroxysteroid dehydrogenase is an
NADP(H)-preferring enzyme. It possesses 11- and 4-fold higher
specificity toward NADP(H) over NAD(H) for oxidation and reduction,
respectively, as demonstrated by kinetic studies. To elucidate the
roles of the amino acids involved in cofactor specificity, we generated
variants by site-directed mutagenesis. The results showed that
introducing a positively charged residue, lysine, at the Ser12 position
increased the enzymes preference for NADP(H) more than 20-fold.
Substitution of the negatively charged residue, aspartic acid, into the
Leu36 position switched the enzymes cofactor preference from NADPH to
NAD with a 220-fold change in the ratio of the specificity toward the
two cofactors in the case of oxidation. This variant dramatically
abolished the enzymes reductase function and stimulated its
dehydrogenase activity, as shown by enzyme activity in intact cells.
The substrate-binding pocket was also studied with four variants:
Ser142Gly, Ser142Cys, His221Ala, and Glu282Ala. The Ser142Gly variant
abolished most of the enzymes oxidation and reduction activities. The
residual reductase activity in vitro is less than
2% that of the wild-type enzyme. However, the Ser142Cys variant
was fully inactive, both as a partially purified protein and in intact
cells. This suggests that the bulky sulfhydryl group of cysteine
entirely disrupted the catalytic triad and that the Ser142 side chain
is important for maintaining the integrity of this triad. His221
variation weakened the apparent affinity for estrone, as demonstrated
by a 30-fold increase in Michaelis-Menten constant, supporting its
important role in substrate binding. This residue may play an important
role in substrate inhibition via the formation of a dead-end
complex. The formerly suggested importance of Glu282 could not be
confirmed.
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INTRODUCTION
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HUMAN ESTROGENIC 17ß-HYDROXYSTEROID
dehydrogenase (17ß-HSD1) (EC 1.1.1.62) is an enzyme implicated in the
biosynthesis and metabolism of steroid hormones. This steroidogenic
enzyme mediates the last step in the formation of active estrogens. The
enzyme principally catalyzes the conversion of the weak E, estrone,
into the most potent E, E2. To a lesser extent, it catalyzes the
interconversion between dehydroepiandrosterone and
androst-5-ene-3ß,17ß-diol, and between progesterone and
20
-hydroxy-4-pregnen-3-one (1, 2). It has been
demonstrated that E2 and androst-5-ene-3ß,17ß-diol stimulate the
growth of mammary tumors (3, 4, 5). E-induced proliferation
is assumed to play a critical role in the promotion of breast cancer
(6, 7), and 17ß-HSD1 plays an important role in the
regulation of in situ E2 production in hormone-
dependent breast cancer (8, 9). Consequently,
17ß-HSD1 represents an important target for breast cancer therapy
(6, 10). Elucidation of the structure-function
relationship of 17ß-HSD1 will facilitate the design of
anti-breast-cancer drugs.
17ß-HSD1 consists of 327 amino acids with a subunit mass of 34,853 Da
(11). It is a homodimer of 68 kDa (12) that
is highly expressed in placenta (13) and ovaries
(14). 17ß-HSD1 is a member of the short-chain
dehydrogenase/reductase (SDR) family (15) that contains
two highly conserved regions. The first region, including a consensus
Gly-X-X-X-Gly-X-Gly motif composed of segments ßA to ßF, is the
classic "Rossmann fold" and associates with cofactor binding
(16). The second region, situated in ßD to ßG,
contains the Tyr-X-X-X-Lys sequence, which has been strictly conserved
during the evolution of the SDR family enzymes and is essential for
enzyme activity (15, 17).
Based on biochemical (2, 18) and structural studies of the
17ß-HSD1-E2-NADP complex (16, 19, 20, 21), 17ß-HSD1
belongs to an NADP(H)-preferring class of enzymes, but it is also able
to bind NAD(H) cofactors in vitro. It has been demonstrated
in a 17ß-HSD1-NADP-equilin complex (21) that the
stabilization of the adenine mononucleotide part of the cofactor is
through contact between the 2'-phosphate of the adenine ribose moiety
and the O
of both Ser11 and Ser12. The positively charged Arg37
interacts with the 2'-phosphate for charge neutralization. In
NADP(H)-preferring enzymes, two conserved positively charged residues
determine the cofactor specificity. One is usually situated before the
second glycine of the Gly-X-X-X-Gly-X-Gly motif, whereas the other is
located at the end of the second ß-strand in the ß
ß folds of
SDR family enzymes. These two positively charged residues are thought
to be able to compensate for the two negative charges of the
2'-phosphate group (22). However, in 17ß-HSD1, only one
positively charged residue, Arg37, is present, with serine substituting
for the other one at position 12. On the other hand, the presence of an
aspartic acid residue at the C terminus of the ßB strand (Leu36 in
17ß-HSD1) in the cofactor-binding motif that electrostatically repels
NADP(H) is a key determining factor in NAD(H) preference
(22). These crystallographic findings have been confirmed
by kinetic studies performed on variants of other SDR family members,
e.g. mouse lung carbonyl reductase (23, 24),
rat hydropteridine reductase (25), and
Drosophila alcohol dehydrogenase (26).
Crystallographic studies of the putative active site of 17ß-HSD1
indicate that the E2 molecule is located in a pocket formed at the
interface of the
G' helix and the loop between ßE and ßF. The
steroid moiety is stabilized by four hydrogen bonds at both ends. The
17-hydroxyl on the E2 D ring can form hydrogen bonds with the hydroxyls
of the conserved Ser142 and Tyr155 residues. These residues thereby act
as an acid/base catalyst toward the 17-hydroxyl atom of the substrate.
The conserved Lys159 is also involved in the proton transfer chain
(16, 19, 20). The 3-hydroxyl on the A ring can form
hydrogen bonds with His221 and Glu282 (19, 21). The
importance of Tyr155, Lys159, and Ser142 has been studied by
site-directed mutagenesis; the variants gave rise to completely
inactive enzymes (27, 28, 29). However, conflicting results
have been published regarding the role of His221 and Glu282 (28, 29).
In this study, we report new structure-function results relating to the
cofactor-binding site with the Ser12Lys and Leu36Asp variants. A single
amino acid substitution, Ser12Lys, significantly increased the
enzymes NADP(H) preference, whereas Leu36Asp changed the enzymes
cofactor preference from NADP(H) to NAD(H). Variation of the four
residues in the active site was carried out, producing Ser142Cys,
Ser142Gly, His221Ala, and Glu282. The kinetics of the variants clearly
demonstrate that the catalysis of the enzyme was significantly
modified. The partially active variant Ser142Gly helped us to further
elucidate the catalytic role of the Tyr155-Lys159-Ser142 triad.
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RESULTS
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Site-Directed Mutagenesis, Variant Expression, and Purification
All of the cDNA mutations were sequenced by the dideoxynucleotide
sequencing method to guarantee the correct mutations. The baculovirus
expression system was used to produce wild type and variants of
17ß-HSD1 in Spodoptera frugiperda (Sf9) cells. Expression
of wild type and variants was confirmed by immunoblotting analysis
using a polyclonal antibody raised against purified human placental
17ß-HSD1. The results of the purified
His6-tagged wild type and variants of 17ß-HSD1
in SDS-PAGE and Western blot analysis are shown in Fig. 1
, A and B. The proteins were purified by
single-step purification using nickel-chelated affinity chromatography
to at least 80% purity as quantified using the PhosphorImager system
(Molecular Dynamics, Inc., Sunnyvale, CA). One variant,
however, Ser142Cys, could not be satisfactorily purified (10% purity;
SDS-PAGE data not shown). Although it is overexpressed in Sf9 cells
(see Fig. 1C
for the Western blot results), the level of expression is
much lower than that in the other variants. The
His6 tag was unable to be effectively cleaved in
the variants and the wild type. This was probably because of the
cleavage site being buried, but it did not affect the activity of the
wild type. According to our previous experience (30) and
published results elsewhere (31, 32, 33, 34),
His6-tagged fusion proteins often retain their
normal biological functions; therefore, we performed the kinetic
studies using the purified His6-tagged
proteins.

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Figure 1. SDS-PAGE (A) and Western Blot (B) of the Purified
17ß-HSD1 Wild-Type and Variants
Samples were run on 12% SDS-PAGE and stained with Coomassie Brilliant
Blue. Two micrograms of each protein was loaded in the well. M,
Low-molecular-mass protein marker; lane 1, wild type purified from
placenta; lanes 27, His6-tagged wild type, Ser12Lys,
Leu36Asp, Ser142Gly, His221Ala, and Glu282Ala. C, Western blot analysis
of Ser142Cys. Lane 1, Purified wild type; lane 2, Ser142Cys in cell
homogenate; lane 3, partially purified Ser142Cys (0.2 µg of each
protein was loaded).
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Effects of Variations in the Cofactor-Binding Site
The steady-state kinetics of wild type 17ß-HSD1, Ser12Lys, and
Leu36Asp variants are summarized in Table 1
. In the presence of a saturating
concentration of cofactors, the apparent Michaelis-Menten constants
(Kms) for E2 using both triphosphate and
diphosphate cofactors for these two variants were not significantly
modified. The apparent Kms for estrone using the
diphosphate cofactor did not change either, but those for estrone using
the triphosphate cofactor increased by about 10-fold for these two
variants (see Discussion). However, the apparent
Kms for the cofactors were significantly
modified. For the Ser12Lys variant, the apparent
Km for the triphosphate cofactors NADP and NADPH
decreased slightly but not significantly, and the apparent
Km for the diphosphate cofactors NAD and NADH
increased about 8-fold. No significant modification of the catalytic
constants (kcat is the turnover numberthe
number of moles of substrate transformed per second per mole of
enzyme) was observed. The enzyme preferences for NADP- and NADPH-linked
reactions [for the ratio of
kcat/KmNADP(H)
over
kcat/KmNAD(H),
see the last column of Table 1
) were about 17- and 26-fold those of the
wild type, respectively. These results indicate that the addition of a
positively charged residue, lysine, at position 12 significantly
decreased the enzymes affinity for NAD(H) and increased the relative
specificity of the enzyme for NADP(H). To verify this, we also
determined the enzyme activity in cultured Sf9 cells. The apparent
activity of Ser12Lys was about 175% that of the wild type for estrone
reduction, whereas it was only about 27% that of the wild type for E2
oxidation after a 30-min reaction (Figs. 2
and 3
). The variation
with this positively charged residue,
therefore, only enhanced the enzymes reductase activity in cells.

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Figure 2. Enzyme Activity of Wild Type, Ser12Lys, and
Leu36Asp Variants in Cultured Sf9 Cells
Mock infection (Sf9 cells without infection) was set as a background
control. A, Estrone reduction; B, E2 oxidation. The results represent
means ± SD of three independent experiments.
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Figure 3. Comparison of the Reduction and Oxidation
Activities of the Wild Type, Ser12Lys, and Leu36Asp Measured in
Cultured Sf9 Cells After a 30-min Reaction
The results represent means ± SD of three independent
experiments.
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The apparent kinetic constants for the Leu36Asp variant changed even
more significantly than those obtained for the Ser12Lys variant. In the
oxidation reaction, the apparent Km for NAD
increased about 6-fold, but that for NADP increased about 2000-fold. No
significant modifications of kcat were
observed. Thus, the Leu36Asp variant dramatically changed from an
11-fold NADP preference to a 20-fold NAD preference, resulting in an
approximately 220-fold modification of the specificity toward the two
cofactors. Similarly, for the reduction reaction, the apparent
Km for NADPH increased 300-fold, and there was no
significant modification for that of NADH. The preference for the
NADH-linked reaction by this variant was 13-fold that of the wild-type
enzyme. The ratio of NADP to NADPH in the cytosol of the liver cells
from a well fed rat is reported to be about 0.014, and that of NAD to
NADH is 700 (35). With such a large
Km value for NADPH in the Leu36Asp variant (257
µM), we expect that the reductase activity of
this variant could be abolished using NADPH. Because the NADH
concentration in the cells would not be high enough to support its
reduction reaction, the variant enzyme would be almost inactive as a
reductase. However, as a dehydrogenase using NAD as the cofactor, its
activity would be enhanced. To verify our prediction, we assayed the
enzyme activity in intact Sf9 cells. It is apparent that the wild-type
enzyme preferentially catalyzes estrone reduction rather than E2
oxidation. However, in the Leu36Asp variant, the estrone reduction
activity was completely abolished, and at the same time, its E2
oxidation activity increased to about 200% of that of the wild type in
Sf9 cells (Figs. 2
and 3
). This variation thus altered the enzymes
preference from NADP(H) to NAD(H) and changed the catalytic designation
of the enzyme from a reductase to a dehydrogenase.
Effects of Variations at the Putative Active Site
Crystallographic studies demonstrate that at the putative active
site of 17ß-HSD1, the substrate can make four hydrogen bonds with
residues in the binding site (19, 21). The 17-hydroxyl on
the D ring forms hydrogen bonds with the hydroxyls of Ser142 and Tyr155
situated at the catalytic end of the steroid-binding cleft. The
3-hydroxyl on the A ring forms bifurcated hydrogen bonds with His221
and Glu282 (Fig. 4
). The key roles of the
conserved Tyr155 and Ser142 in maintaining enzyme activity have been
demonstrated (27, 28, 29), but the roles of His221 and Glu282
need to be investigated further. In our study, we constructed
Ser142Cys, Ser142Gly, His221Ala, and Glu282Ala variants. The Ser142Cys
was inactive both in a partially purified enzyme form and in intact Sf9
cells. The activity of the Ser142Gly variant in both reduction and
oxidation reactions was significantly decreased. The reduction activity
was less than 2% that of the wild type. However, in intact cells under
our experimental conditions, it still retained about 27% of the wild
type enzyme activity for estrone reduction (Table 2
and Fig. 5
). The discrepancies in activity between
purified enzyme and intact cells may be caused by instabilities in the
variant after cell disruption.

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Figure 4. Active Site Stereochemistry of the E2-Bound
17ß-HSD1
Hydrogen bonding interactions are represented by dotted
spheres. The water molecules are in green. For
E2 and NADP, the atom coloring is as follows: carbon atoms are in
white, oxygen atoms are in red,
phosphorus atoms are in yellow, and nitrogen atoms are
in blue. The NADP molecule is modeled in our previous
crystallographic study (19 ).
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Figure 5. Comparison of the Reduction and Oxidation
Activities of the Wild Type, Ser142Cys, Ser142Gly, His221Ala, and
Glu282Ala in Cultured Sf9 Cells After a 30-Min Reaction
The results represent means ± SD of three independent
experiments.
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The kinetic constants of the His221Ala variant were altered from those
of the wild type. The apparent Km of estrone
reduction using NADPH as the cofactor increased 30-fold, and the
apparent specificity
(kcat/Km) decreased
about 65-fold. The apparent Km of E2 oxidation
using NADP as the cofactor increased 4-fold, and the apparent
specificity decreased 5-fold (Table 2
). The kinetic constants changed
more significantly during the estrone reduction using NADPH as the
cofactor (Table 2
), but in intact cells, the estrone reduction activity
was not significantly altered (Fig. 5
) (for explanation, see
Discussion). However, the variant Glu282Ala did not show any
significant modification either in vitro or in intact Sf9
cells.
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DISCUSSION
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17ß-HSD1 was once classified as an NAD(H)-preferring enzyme
based on its amino acid sequence alignment (22). Now,
based on kinetic studies (2, 18), it is evident that
in vitro this enzyme is able to use both NAD(H) and NADP(H)
as cofactors. However, in transfected intact HEK293 cells (human
embryonic kidney cells), it is an NADP(H)-preferring enzyme that almost
unidirectionally catalyzes estrone reduction (2). One
important specific feature of 17ß-HSD1 is reflected by the ratio of
its apparent specificity for NADP(H) and NAD(H). The apparent
specificity of the enzyme to the triphosphate cofactors is no more than
12-fold higher compared with that of the diphosphate cofactors. This is
in marked contrast to other NADP(H)-preferring enzymes, such as mouse
lung carbonyl reductase (23, 24), or NAD(H)-preferring
enzymes, such as Drosophila alcohol dehydrogenase
(26), in the SDR family, or the NADP(H)-preferring enzymes
from other families, such as NADPH-cytochrome P450 oxidoreductase
(36) and human aldose reductase (37). These
enzymes display hundreds- to thousands-fold higher specificity for one
cofactor than the other.
With regard to the enzyme structure, two positively charged residues
for the NADP(H)-preferring enzyme are thought to be important for
interaction with the negatively charged ribose 2'-phosphate of NADP(H)
(22). One positively charged residue located in the
cofactor-binding motif is lacking in 17ß-HSD1, replaced by a serine
at residue 12. Compared with NAD(H)-preferring enzymes, the 17ß-HSD1
also lacks a negatively charged residue (usually an aspartic acid
residue) at residue 36 of the enzyme. This aspartic acid residue is
shown to form a bifurcated hydrogen bond to both the 2'- and
3'-hydroxyl groups of the ribose moiety of NAD(H) to stabilize the
cofactor NAD(H). However, it can result in steric hindrance and/or
electrostatic repulsion toward the 2'-phosphate of NADP(H)
(22) and is thought to be characteristic of
NAD(H)-preferring enzymes (22, 26). Using molecular
modeling with energy minimization based on the crystallographic results
of Mazza et al. (38), we have shown the
cofactor-enzyme interaction (Fig. 6
).
This unfavorable interaction between the side chain of the Asp36 and
the ribose phosphate of NADP is also demonstrated in our homology
modeling result (Fig. 6B
). The charge neutralization resulting from
Arg37 interaction with the O2'-adenine ribose
phosphate has been abolished by the introduction of the Asp36, which
forms an unfavorable interaction and repels the two oxygen atoms of the
adenine ribose. By having such a structural arrangement in the cofactor
binding site, 17ß-HSD1 is able to accommodate both NADP(H) and
NAD(H). Of interest, compared with 17ß-HSD1, in which the Leu36Asp
variation selectively abolishes the enzymes NADP(H) preference while
keeping the enzymes NAD(H) preference, is the observation of
reverse-sense variation reported in the Drosophila alcohol
dehydrogenase (26). In the latter case, the substitution
of an Asp38 to Asn increases the enzymes NADP(H) preference but does
not interfere with the enzymes affinity for NAD(H). Thus, the enzyme
can use both NAD(H) and NADP(H) as cofactors in vitro, a
situation observed in 17ß-HSD1.

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Figure 6. Crystal Structures and the Results of Homology
Modeling with Energy Minimization of the Cofactor-Binding Site for
17ß-HSD1
A, Structure of the NADP-binding site from the E2-NADP-17ß-HSD
structure at 1.9 Å resolution (38 ). The figure shows the
position of residues Ser12, Leu36, and Arg37 relative to NADP
2'-phosphate. The green dots represent the potential
hydrogen bonds between the side chain of Arg37 and 2'-phosphate of the
adenine ribose and between the side chain of Ser12 and the O2'
of the pyrophosphate moiety. B, Model of the NADP-binding site with the
Leu36Asp variation obtained by homology modeling with energy
minimization on the structure shown in A. Pink dots show
potentially unfavorable interactions between the Asp36 side chain
carboxyl and two oxygen atoms of the NADP adenine ribose phosphate. For
both panels, oxygen atoms are in red, nitrogen atoms are
in blue, sulfate atoms are in yellow, and
carbon atoms are in white.
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In the absence of this negatively charged aspartic residue, the
presence of at least one positively charged residue in the cofactor
binding site to interact with the 2'-phosphate may be important for
maintaining NADP(H) specificity. In a protein sequence alignment study
of a group of known NADP(H)-preferring enzymes in the SDR family, 36 of
46 possessed at least one positively charged residue at either of the
two positions mentioned above (38). In the current study,
we observed the alterations caused by the variants in intact Sf9 cells,
which may more closely reflect the physiological state. 17ß-HSD1 has
the potential to act both as a dehydrogenase and a reductase in
vitro. However, in vivo, where NADPH and NAD are
present simultaneously at high enough concentrations to support
oxidation and reduction reactions, NADPH, with a higher affinity for
the enzyme, would strongly occupy the cofactor-binding site and drive
the catalytic reaction toward the reduction direction. The cofactor
specificity of the enzyme and the cofactor concentration in the tissues
in which the enzyme is principally located determines the reaction
direction. Our study of the variants Ser12Lys and Leu36Asp also
supports the fact that in intact cells an enzyme using NADP(H) as a
cofactor principally works as a reductase and an enzyme using NAD(H) as
a cofactor plays the role of a dehydrogenase (2, 39).
The ability of Ser142 together with the two highly conserved residues
Tyr155 and Lys159 to form a catalytic triad in 17ß-HSD1 and in mouse
lung carbonyl reductase of the SDR family has been demonstrated
(16, 22). In prokaryotic 3ß/17ß-HSD, the
serine-to-alanine substitution at position 138 resulted in more than
99.9% loss of enzyme activity, and its kinetic constants could not be
determined (40). However, the serine-to-threonine
substitution at this position resulted in a fully active enzyme,
because threonine has a hydroxyl group in the side chain
(40). The fact that the Ser142Ala variation resulted in an
almost inactive enzyme in both cell homogenates and intact cells
has been reported for human 17ß-HSD1 (29). The present
study, together with the former reports, further demonstrates the
importance of the hydrogen bond between the serine side chain and the
17-hydroxyl of the substrate. An appropriate side chain with a hydroxyl
group in the residue is essential for catalytic activity. In the
Ser142Cys variant, cysteine has a molecular composition very similar to
serine except that the side chain hydroxyl is substituted by a
sulfhydryl. The bulky sulfhydryl group is unable to form a hydrogen
bond with the 17-hydroxyl and may further disrupt the hydrogen bond
between the 17-hydroxyl and Tyr155 by imposing steric hindrance, thus
destroying the catalytic chain. In the case of the Ser142Gly variant,
the smallest residue lacking both a side chain and a hydroxyl group is
unable to form a hydrogen bond with the 17-hydroxyl, thereby
significantly decreasing the enzyme specificity. Because the hydrogen
bond between Tyr155 and the 17-hydroxyl may not have been disrupted,
catalytic function is partially retained. This supports the proposed
reduction mechanism in which Ser142 itself does not donate the proton
to 17-hydroxyl but is able to stabilize the unprotonated state of
Tyr155 by sharing its proton with Tyr155 (20).
As shown in the present study, the residue His221 at the 3-hydroxyl
side of the substrate is not as critical as Ser142 and Tyr155 for
maintaining enzyme activity, although the kinetic constants are altered
and the activity in intact cells is partly modified (oxidation
reaction). The results that we obtained follow the same trend as those
reported by Puranen et al. (29): the reductase
activity of His221Ala is maintained in intact cells, which is different
from the results in vitro. Such a discrepancy was explained
as the interaction between the 3-hydroxy group of the substrate and the
His221 side chain occurring only in vitro and not in
vivo (29). We suggest that this is caused by the
elimination of the enzymes substrate inhibition phenomenon by using
NADPH as cofactor in this variant. Substrate inhibition is a phenomenon
observed in the wild-type 17ß-HSD1, and it was recently reported by
our laboratory (41). Substrate inhibition is produced in
most natural hydroxyacid dehydrogenases by the formation of an abortive
enzyme-NAD(P)-keto acid complex (dead-end complex) in the presence of a
high concentration of keto acids (42). Substrate
inhibition occurs only when using estrone as the substrate and NADPH as
the cofactor at estrone concentrations greater than 0.2
µM in 17ß-HSD1. The substrate
inhibition phenomenon in 17ß-HSD1 may suggest the presence of a self-
protective mechanism by limiting the effects of increasing
intracellular estrone levels in vivo (41). In
the presence of high keto-substrate concentration (10
µM), the variant His221Ala could indeed show
higher activity than the wild-type enzyme in the reduction in intact
cells as a result of the decrease of substrate affinity and hence the
elimination of the substrate inhibition. Thus, His221 may play an
important role in the control of substrate accessibility and product
release. It may participate in the regulatory mechanism of E2 formation
together with other residues, such as the two charged residues Ser12
and Leu36 at the cofactor-binding site. Within the substrate
concentration range used in our experiments, we observed that the
Km for estrone using NADPH as a cofactor
increased 10-fold, whereas the substrate inhibition phenomenon
disappeared in the Ser12Lys and Leu36Asp variants. Glu282 is
anticipated to play the same important role as His221Ala, because it
might form a hydrogen bond with the O3 of the
substrate, as suggested by the E2 complex structure. However, based on
our kinetic results, we are unable to prove its importance in the
catalysis of the enzyme.
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MATERIALS AND METHODS
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Materials
Restriction endonucleases and modifying enzymes were purchased
from Amersham Pharmacia Biotech (Uppsala, Sweden) and
Roche Molecular Biochemicals (Indianapolis, IN).
Taq DNA polymerase and Pfu DNA polymerase were from
Perkin-Elmer Corp. (Branchburg, NJ) and
Stratagene (La Jolla, CA), respectively. Sf9 cells,
baculovirus expression system (Autographa californica
Nuclear Polyhedrosis Virus, AcMNPV linear transfection module), and
transfer vector pVL1393 were from Invitrogen (Carlsbad,
CA). Transfer vector pFastBac- and DH10Bac-competent cells were from
Life Technologies, Inc. (Grand Island, NY). The QuikChange
site-directed mutagenesis kit was from Stratagene. All
media and supplements used for cell culture were from Life Technologies, Inc. NAD(H), NADP(H), phenylmethylsulfonyl
fluoride, dithiothreitol, E2, and estrone were from Sigma-Aldrich Corp. Canada Ltd. (Oakville, Ontario, Canada).
[14C]Estrone and
[14C]E2 (56.6 mCi/mmol) were from
DuPont-New England Nuclear Life Science Products (Boston,
MA). All other reagents were of the highest grade purity and purchased
from Sigma-Aldrich Corp.
Site-Directed Mutagenesis of Human 17ß-HSD1
Human 17ß-HSD1 Leu36Asp, Ser142Cys, and Ser142Gly point
mutations were carried out by PCR (43). The 17ß-HSD1
cDNA previously isolated and characterized in our laboratory
(11) was used as a template for generating variants. PCR
fragments were subcloned into the baculovirus transfer vector pVL1393.
Ser12Lys, His221Ala, and Glu282Ala point mutations were carried out in
the vector pFastBac1 using the QuikChange site-directed mutagenesis kit
according to the manufacturers instructions. Table 3
shows the six different substitutions
created in human 17ß-HSD1 and the oligonucleotide primers used for
the mutagenesis. To facilitate protein purification, a nucleotide
sequence coding for six consecutive histidine residues followed by a
factor Xa cleavage site was added at the 5' terminus of the 17ß-HSD1
wild type and all of the variants by PCR. To ensure that the expected
substitutions had occurred, cDNA sequences of the mutated 17ß-HSD1
were verified by dideoxynucleotide sequencing [Big Dye Terminator
Cycle Sequencing Ready Reaction kit (PE Applied Biosystems, Foster City, CA) 373 sequencer with XL
Upgrade].
Production of Recombinant Baculovirus
Generation of the Leu36Asp, Ser142Cys, and Ser142Gly mutated
17ß-HSD1 recombinant baculoviruses was achieved using the AcMNPv
linear transfection module. For more convenient and efficient
overproduction of the mutated proteins, we switched our transfer vector
and cotransfection system from pVL1393 and the AcMNPv linear
transfection module to the Bac-to-Bac system (Life Technologies, Inc.). This system saved time by omitting the
plaque purification step in insect cells while still achieving the same
expression efficiency as the former system (44).
Generation of native and Ser12Lys, His221Ala, and Glu282Ala mutated
17ß-HSD1 and all of the variants as well as wild-type 17ß-HSD1 with
His6-tagged recombinant baculovirus was achieved
using the Bac-to-Bac baculovirus expression system according to the
manufacturers instructions. Five microliters (
5 µg) of
recombinant bacmid DNA and 6 µl of cell FECTIN were mixed to infect
the monolayers of Sf9 cells at 27 C for 45 d. The supernatants were
collected as P1 recombinant baculovirus stocks.
Expression of Wild-Type and Mutated 17ß-HSD1 in Sf9 Insect
Cells
Production of 17ß-HSD1 from Sf9 cells was performed as
described by Breton et al. (45). Recombinant
baculovirus previously amplified and titered was used to infect Sf9
cells (1.82 x 106/ml) at a multiplicity
of infection of 10. The infection was performed at 27 C. Cells were
harvested 60 h after infection by 10 min of centrifugation at
1,000 x g, washed once with PBS, pelleted again, and
kept at -80 C for later use.
Protein Purification by Nickel-Chelated Affinity
Chromatography
The purification steps were carried out at 4 C unless otherwise
specified. Before purification, cell pellets from about 6 x
108 cells were suspended in 2040 ml of buffer A
(40 mM Tris, pH 8.0, 100 mM NaCl, 20%
glycerol, 0.4 mM phenylmethylsulfonyl fluoride, and 4
mM imidazole). Cells were disrupted by sonication (5
x 0.5 min at 0.5-min intervals with output control at 2.5) and
centrifuged at 110,000 x g for 30 min. The AKTA
Explorer 100 Air System (Amersham Pharmacia Biotech, Baie
dUrfé, Québec, Canada) was used to purify the
proteins. The supernatants were collected, loaded onto a
nickel-nitrilotriacetic acid agarose superflow column
(QIAGEN Inc., Mississauga, Ontario, Canada),
preequilibrated with buffer A, washed, and eluted with the linear
gradient of imidazole in the same buffer at a flow rate of 1 ml/min.
Most of the specific protein was eluted at imidazole concentrations of
7090 mM. The fractions containing 17ß-HSD1
were identified by activity assay, electrophoresis, and immunoblotting.
The peak fractions were concentrated by Centricon-30 (Amicon, Beverly,
MA).
Enzyme Activity in Cultured Cells
Enzyme activity in cultured cells was assessed by plating cells
in six-well plates at a density of 1.2 x
106/well. Cells were left to attach for 1 h
before viral infection. A mock infection was set up as a negative
control. The medium was removed after a 50-h incubation at 27 C, and 2
ml of serum-free TNM-FH medium with 10 µM
[14C]estrone or [14C]E2
(2.8 µCi/µmol, 20 nmol/well) was added to each well. Reactions were
carried out at room temperature. At various times (3, 10, 15, 30, and
45 min for reduction; 10, 15, 30, 60, and 120 min for oxidation),
aliquots of the media were transferred to tubes containing 3 vol of
cold diethyl ether. The steroids were extracted with ethanol on dry ice
and dried by evaporation. They were then dissolved in dichloromethane,
subjected to TLC, separated by toluene-acetone (4:1, vol/vol), and
quantified with a Storm 860 Laser Scanner (Molecular Dynamics Inc., Sunnyvale, CA) with ImageQuant software.
Steady-State Kinetics
Kinetic constants were determined using purified
His6-tagged proteins. A radioactive assay was
used for determination of the kinetic constants. The reaction mixture
contained 50 mM potassium phosphate, pH 7.4, and 50 µg/ml
BSA. Kinetic constants of estrone were determined by fixing NADH and
NADPH concentration at 1.0 and 0.1 mM respectively (except
for Leu36Asp, which used 1.5 mM NADPH);
[14C]estrone
concentrations ranged from 0.0075 µM. Kinetic constants
of E2 were determined by fixing NAD concentration at 1 mM
and NADP concentration at 0.1 mM (except for Leu36Asp,
which used 5 mM NADP); [14C]E2
concentrations ranged from 0.01425 µM. Kinetic
constants of cofactors NAD(H) and NADP(H) were achieved by fixing
substrate [14C]E2 concentration at 25
µM [14C]estrone concentration at
10 µM; cofactor concentrations ranged from 0.15000
µM. The initial velocity was measured with less than 5%
substrate conversion. Reactions were carried out at 23 C and stopped at
four different times by cooling 0.5 ml of reaction mixture in 4 C
diethyl ether. The steroids were extracted and quantified as described
above. For the determination of all of the kinetic constants, at least
three independent experiments were performed. The
Kms for the substrates and for the cofactors were
determined by Lineweaver-Burk plots. One unit of enzyme activity is
defined as the amount of enzyme that catalyzes the formation of 1
µmol of product in 1 min. The kcat values
were calculated from the maximum initial velocity values with the
homodimer molecular mass of 72 kDa [68 kDa of 17ß-HSD1
(12) plus 4 kDa of His tag].
Protein Concentration Determination
Protein concentrations of the cell homogenate and enzyme
preparations during the purification were determined using the Bradford
reagent (Bio-Rad Laboratories, Inc., Hercules, CA). The
interference of imidazole was corrected using the same buffer in the
absence of the protein.
Gel Electrophoresis and Western Blot Analysis
Wild-type and mutated 17ß-HSD1 were submitted to gel
electrophoresis on a homogeneous 12% gel using the Bio-Rad Laboratories, Inc. Mini-PROTEAN II apparatus. Gels were
electroblotted onto nitrocellulose membranes for Western blot analysis.
Blots were probed with a rabbit polyclonal antibody raised against
purified human placental 17ß-HSD1 as the primary antibody and
horseradish peroxidase-conjugated donkey antirabbit polyclonal antibody
(Amersham Pharmacia Biotech, Little Chalfont,
Buckinghamshire, UK) as the secondary antibody. The immunoreactive
blots were detected with enhanced chemiluminescence reagents
(DuPont-New England Nuclear Life Science Products)
and exposed to x-ray film (Eastman Kodak Co., Rochester,
NY).
Homology Modeling
The models for the Leu36Asp mutants were obtained by homology
modeling using the Swiss-PdbViewer platform V3.6b3 (46)
and were minimized to correct bad contacts using the GROMOS 96 force
field (47). We have introduced a Leu36ASP variation on the
NADP-E217ß-HSD1 structure (38).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. F. Labrie for his interest in this work. We also
thank Dr. P. Rehse for the critical reading and M. Losier for the
editing of the manuscript.
 |
FOOTNOTES
|
---|
This work was supported by the Medical Research Council of Canada.
Y.-W.H. was supported by a studentship from the Fonds pour la Formation
de Chercheur et lAide à la Recherche/Fonds de la Recherche en
Santé du Québec.
1 These authors made the same contribution for variant
construction. 
2 Current address: Agriculture et Agroalimentaire Canada, Station de
Recherchers sur les Sols et les Grandes Cultures, Sainte-Foy,
Québec, Canada. 
Abbreviations: E2, Estradiol; E, estrogen; E1, estrone;
17ß-HSD1, human estrogenic 17ß-hydroxysteroid dehydrogenase;
Km, Michaelis-Menten constant; NAD, nicotinamide
adenine dinucleotide; NADH, reduced nicotinamide adenine
dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; NADPH,
reduced nicotinamide adenine dinucleotide phosphate; SDR,
short-chain dehydrogenase/reductase; Sf9, Spodoptera
frugiperda.
Received for publication October 17, 2000.
Accepted for publication July 30, 2001.
 |
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