From the Laboratoire de Cristallographie et Cristallogenèse
des Protéines, Institut de Biologie Structurale J.-P. Ebel,
CEA-CNRS, 41, avenue des Martyrs,
F-38027 Grenoble cedex, France
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INTRODUCTION |
The 17
-estradiol (E2) is known to promote the
genesis and development of human breast cancers (1, 2). Its presence in
tumor cells comes from in situ synthesis (3), and its
concentration is significantly increased in malignant breast tissues
(4-6). Type 1 17
-hydroxysteroid dehydrogenase
(17
-HSD1)1 catalyzes the
reversible transformation of estrone (E1) into the
biologically active estradiol (E2) (7). Thus, preventing the formation of E2 by a specific inhibition of 17
-HSD1
activity should be of paramount importance for cancer therapy.
The 17
-HSD1 (Mr = 34,900, 327 residues) is
active as a homodimer (8) and requires a dinucleotide cofactor (either
NADP+/NADPH or NAD+/NADH) to transform
estrogens. Amino acid sequence alignments revealed that it belongs to
the short chain dehydrogenase reductase family (SDR) (9-11). A
consensus sequence Tyr-Xaa-Xaa-Xaa-Lys (Tyr155 and
Lys159 in 17
-HSD1) as well as a generally conserved
serine residue (Ser142 in 17
-HSD1) characterize this
protein family. A catalytic role for the conserved tyrosine and lysine
residues has been suggested by site-directed mutagenesis experiments
(12-16), and subsequent crystallographic studies (17-26) have
confirmed the importance of the consensus triad. In the
17
-HSD1·E2·NADP+ complex (25),
Tyr155, Ser142, and the E2
O17 atom form a triangular hydrogen-bonding arrangement
that may result in easier deprotonation of the putatively reactive
tyrosine. Lys159 participates in the NAD(P)+
stabilization by establishing hydrogen bonds with O2' and
O3' of the nicotinamide ribose.
Based on amino acid sequence alignments, 17
-HSD1 was thought to
belong to the group of NAD(H)-preferring enzymes (22). However,
biochemical studies (7) and the structure of the
17
-HSD1·E2·NADP+ ternary complex (25)
have shown that 17
-HSD1 is able to bind both NAD(H) and NADP(H).
17
-HSD1 appears to be unique among the SDR family because it lacks
both the aspartic acid residue at position 36 (Leu36 in
17
-HSD1), characteristic of NAD(H) preferring enzymes, and the basic
residue located in the consensus sequence of the dinucleotide binding
motif Gly-Xaa-Xaa-Xaa-Gly-Xaa-Gly (which is replaced by Ser12 in 17
-HSD1). This motif forms an ionic interaction
with the ribose 2'-phosphate and is characteristic of
NADP(H)-preferring enzymes.
His221, first identified by affinity labeling studies
(27-29), was thought to be involved in the specific binding of the
steroid. Indeed, the construction of an H221A mutant led to an enzyme
displaying a higher Km and lower
Vmax relative to the wild type (16).
Furthermore, the crystal structure of the enzyme complexed with
E2 (25, 26) revealed that His221 is directly
involved in the specific binding of the steroid.
Here, we report the construction of H221L and H221Q mutants, their
characterization by enzymatic assays, their crystallization, and the
determination of the structures of the binary complexes H221Q·E2 and H221L·NAD+ and the ternary
complex H221L·NADP+·E2 at 2.7, 3.0, and 2.7 Å resolution, respectively. We show for the first time a well ordered
conformation for the 191-199 loop and speculate about its role in
cofactor binding and hydride transfer. Moreover, the specificity of the
enzyme for estrogens is reassessed.
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EXPERIMENTAL PROCEDURES |
Materials--
Spodoptera frugiperda, purified AcNPV
DNA (Autographa Californica nuclear polyhedrosis virus), and
transfer vector pVL1393 were purchased from Invitrogen Corporation;
Grace's insect cell culture medium, yeastolate, lactalbumin
hydrolysate, fetal bovine serum, and restriction enzymes were from Life
Technologies Inc.; NAD+, NADP+, estradiol, and
estrone were from Sigma; and Blue-Sepharose CL-6B was from Amersham
Pharmacia Biotech.
Cell Culture and Virus--
The TNM-FH medium was prepared from
Grace's medium by the addition of 3.3 g/liter yeastolate and 3.3 g/liter lactalbumin hydrolysate. The Sf9 cells were grown as
monolayers at 28 °C in TNM-FH medium supplemented with 10% fetal
bovine serum. Cells were infected with virus at a multiplicity of
infection of 0.1-1 plaque-forming unit to produce virus stocks or at
multiplicity of infection >10 for maximal protein expression. Cells
were harvested 60 h after infection.
Site-directed Mutagenesis--
The two mutants H221Q and H221L
were constructed with one round of polymerase chain reaction made on
the pVL/17
-HSD transfer vector previously constructed for 17
-HSD1
overexpression in baculovirus (30). These mutations use two primers.
The first one is located on the cDNA, upstream of a
PstI unique site (5'-GTAGTAGGGACTGTGCGG-3'). The second one
introduces the mutation. For H221Q and H221L, it overlaps the codon to
be mutated and a NruI unique site
(5'-GCCGCCTCGCGAAAGACTTGCTTGCTTTGGGCGAGG-3' and
5'-GCCGCCTCGCGAAAGACTTGCTTGCTGAGGGCGAGG-3', respectively). Amplified fragments and pVL/17
-HSD1 were digested with
NruI and PstI. The mutated fragments were then
cloned instead of the nonmutated one. Mutations were checked by
dideoxynucleotide sequencing, and mutated transfer vectors were
cotransfected in Sf9 cells with the wild-type AcNPV virus
following the protocol described by Invitrogen. Recombinant viruses
were purified by plating.
Protein Purification and Enzyme Assay--
The purification of
mutant proteins was performed as described previously for the wild-type
protein (30, 25). The only modification concerned the H221L mutant
which was purified at pH 7.0 instead of pH 7.5. The activities of the
wild-type and the mutated 17
-HSD1s were measured as described by
Langer and Engel (31) with few modifications. The enzymes were assayed by spectrophotometric measurement of the concentration changes of NADPH
at 340 nm. Reactions were run in 50 mM
NaHCO3/Na2CO3 buffer, pH 9.2, for
the oxidation and 50 mM
KH2PO4/K2HPO4 buffer, pH 5.8, for the reduction, both containing 0.5-16 µM
steroid (estradiol or estrone, respectively). In both cases, 10 µl of
enzyme preparation (from 55 to 90 µg/ml) were added to 500 µl of
buffer. Reactions were initiated by the addition of 5 µl of a 10 mM NADP+ or NADPH solution, and the absorbance
variations were measured against a blank containing all components
except steroid and coenzyme. Controls containing all components except
steroid were also run. Reactions were followed from 30 s to 3 min
after addition of NADP+ on a Shimadzu UV-160A
spectrophotometer. For NADP+ reduction measurements, the
same protocol was used except that the estradiol concentration was
fixed to 25 µM while NADP+ concentrations
varied from 0.05 up to 5 mM. Velocities were calculated from the slopes of the zero order portion of the kinetics obtained and
were corrected for the control absorbance. The enzymatic activity was
calculated as described previously (8) and defined in units per
milliliter (taking a micromole per min as a unit). Lineweaver-Burk plots were used to determine the Km and the
Vmax values. Protein concentrations of enzyme
preparations were measured using a micro BCA protein reagent kit from
Pierce. The catalytic activity parameters are presented in Tables IV
and V.
Crystallization--
All recombinant mutant enzymes were
concentrated to 5 mg/ml, in a buffer containing 40 mM Tris,
pH 7.5, 1 mM EDTA, 0.2 mM DTT, 20% glycerol,
and 2 mM
-octylglucoside and crystallized at room
temperature. The H221Q mutant was crystallized in conditions similar to
those of the wild-type enzyme (100 mM HEPES, pH 7.0, 100 mM MgCl2, 0.5 mM E2,
2-4% propanediol, 30% polyethylene glycol 4000) (25, 32); crystals
appeared after 6 days. The H221L mutant was crystallized in 100 mM sodium phosphate buffer, pH 6.0-6.3, 1 mM
NAD(P)+, 100 mM NaCl and 2.2-4.4
mM decyl-
-D-maltoside or 9-18
mM octyl-
-D-thioglucopyranoside and 2-2.4
M ammonium sulfate as precipitant. Space groups and unit
cell dimensions of these crystals are summarized in Table I.
The H221L crystals used were incubated (from a few minutes up to one
night) in a buffer containing 100 mM HEPES, pH 6.5, 1 mM NADP+, 100 mM NaCl, 30% PEG
4000 with and without 0.5 mM E2 to obtain both
the ternary and the binary complex. Crystals were then flash-cooled under a nitrogen gas stream at
150 °C, transferred into liquid propane at
160 °C, and conserved in solid propane at
196 °C until data collection was carried out at a synchrotron radiation source.
Data Collection and Processing--
A data set of the H221Q
mutant was collected at room temperature on the in-house
Siemens/Xentronics area detector X1000 system, mounted on a Rigaku
RU200 rotating-anode-x-ray generator. Two data sets of the H221L mutant
were collected at low temperature (
150 °C) on the D2AM
French-Collaborative Research Group synchrotron beam line at the
European Synchrotron Radiation Facility (ESRF, Grenoble), using an
XRII-CCD detector (33). Data were processed with XENGEN (34) for the
former and with a modified version of XDS (35, 36) for the others. Data
collection statistics are summarized in
Table I.
Structure Determination--
For the H221Q mutant, a rigid body
refinement with the model of the wild-type 17
-HSD1 was sufficient to
place it correctly in the unit cell. The H221L mutant structure was
solved by molecular replacement methods with the wild-type 17
-HSD1
as a search model using AMoRe (37). Four molecules per asymmetric unit
were found. The mutants structures were refined with X-PLOR (Version
3.1) (38) and REFMAC (CCP4 Suite of Programs) (39), and model
corrections were made with O (40). The refinement statistics are
presented in Table II. The coordinates
have been deposited with the Protein Data Bank (codes: 1FDU for the
H221L·E2·NADP+ complex, 1FDV, for the
H221L·NAD+ complex, and 1FDW for the
H221Q·E2 complex).
 |
RESULTS |
Two different crystalline complexes were obtained with the H221L
mutant: the first one by co-crystallizing the protein with NADP+ and diffusing E2 in the crystal, and the
second one by crystallizing the protein in the presence of
NAD+. Both complexes crystallize in the monoclinic
P21 space group (Table I) with four molecules in
the asymmetric unit corresponding to two biologically active dimers
(Fig. 1), here named mA/mB and mC/mD,
respectively.

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Fig. 1.
The
H221L·E2·NADP+ complex. The
non-crystallographic 2-fold axis is shown between the two monomers. The
two Rossmann-fold motifs ( A- B- B- C- C- D- D) are
depicted in light pink and light blue.
Lys195, Arg37, and Ser11
(yellow) interact with the NADP+ 2'-phosphate.
Arg37 and Ser11 belong to Rossmann-fold loops
while Lys195 is located in a variable domain. (This figure
was generated with Molscript (48) and Render (49, 50).)
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In the H221L·NADP+·E2 ternary complex, the
electron density is very well defined all along the polypeptide chain,
including the 191-199 loop that was disordered in the wild-type
structure (Fig. 2). The observed
conformation for this loop is completely different from that proposed
by Ghosh et al. (24) but the 190-192 segment
conformation is similar to that of residues 184-186 in mouse lung
carbonyl reductase (MLCR) (22) (Fig. 3).
The electron density map is also very well defined for the steroid
and cofactor (Fig. 4). As a result,
NADP+ and estradiol have been modeled with full occupancies
in the four subunits.

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Fig. 2.
Stereoscopic view of the (2Fo-Fc) electron
density map around the 189-200 loop of the monomer mC of the
H221L·E2·NADP+ complex. The map,
computed with 2.7 Å resolution data, is contoured at the 1 level.
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Fig. 3.
Superposition of the 187-209 loop of the
wild-type 17 -HSD1 model (1bhs (24)) (medium gray) and
the monomer mC of the H221L·E2·NAD+ complex
(black) with the 182-203 loop of the MLCR model (1cyd
(22)) (light gray). Phe192 of the H221L
mutant and Met186 of MLCR superpose very well and make
extensive hydrophobic contacts with the nicotinamide ring.
Lys195 of the H221L model interacts with the 2'-phosphate
while no equivalent residue is found for MLCR.
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Fig. 4.
Stereoscopic view of the (2Fo-Fc) electron
density map around the NADP+, the residues
Ser11 and Arg37 and the 190-196 loop of the
monomer mC of the H221L·E2·NADP+
complex. The map, computed with 2.7 Å resolution data, is
contoured at the 1 level.
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In the H221L·NAD+ binary complex, the electron density
for NAD+ and the 191-200 loop is well defined for monomers
mA and mC but discontinuous for monomers mD and mB. Consequently, the
191-199 loop was not included in the models of these two monomers.
Since this site is known to have Michaelian kinetics, we do not
understand the observed difference in NAD+ occupancy. It
may be due to subtle packing effects difficult to characterize at
this resolution.
Both NADP+ and NAD+ bind in the same extended
conformation already observed for the wild-type enzyme: the cofactor
points toward the active site of the enzyme with the nicotinamide ring
in the syn conformation and the adenine in the
anti conformation (25).
The structure of the H221Q·E2 binary complex displays two
conformations for Gln221. In one of these, the amide group
of the Gln221 forms a hydrogen bond with the steroid
O17 atom; whereas in the other, Gln221 is
oriented toward the solvent. The double conformation of the Gln221 may be a consequence of the partial occupation of
the steroid-binding site, as it was already suggested in the case of
the wild-type enzyme (25). No electron density was found for the
cofactor.
Having a complete model of the cofactor binding site allows for a full
description of the NAD(P)+/protein interactions. Some of
these interactions are common to NADP+ and
NAD+, and most of them were present in the wild-type
ternary complex (25). However, due to the disorder in the neighborhood
of the NADP+ binding site in the latter structure, two
major interactions were not observed: the extensive hydrophobic contact
between the nicotinamide moiety and the Phe192 side chain,
and the charge compensation of the dinucleotide 2'-phosphate through
salt bridges with Arg37 and Lys195 side chains
(Figs. 4 and 5). In addition, the
2'-phosphate is further stabilized by a hydrogen bond with
Ser11 O
. In the NAD+·H221L
complex, the binding pocket of the ribose 2'-phosphate of monomers mA
and mC is occupied by a sulfate ion, presumably coming from the
crystallization solution. This ion is bound to the
protein·NAD+ complex through a hydrogen bond network
involving 2'-OH adenine ribose, Lys195, Arg37,
Ser11, and Thr41 side chains. This phenomenon
has already been observed in glutathione reductase where an inorganic
phosphate ion substitutes for the missing 2'-phosphate group when
NAD+ is bound (41). In both NAD+ and
NADP+ complexes, the NH3+ group of
Lys195 interacts with the O1A of the pyrophosphate.

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Fig. 5.
The NADP+ environment. The
191-199 loop is represented in black, and NADP+
and estradiol are in medium gray. The 2'-phosphate group is
stabilized by three interactions (dashed lines): two salt
bridges with Arg37 and Lys195 and a hydrogen
bond with Ser11. Phe192 protects the
nicotinamide from solvent by an extensive hydrophobic contact. The
catalytic site formed by Ser142, Tyr155, the
steroid O17 atom, and nicotinamide are also represented.
Dashed lines represent the triangular hydrogen bond
arrangement between Ser142, Tyr155, and
O17.
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The 191-199 loop, located between the
F sheet and the
G helix,
seems to be predominantly stabilized by its interactions with the
dinucleotide, particularly by the salt bridge between Lys195 and the 2'-phosphate. In the
H221L·NAD+ complex, where a sulfate ion replaces the
2'-phosphate, the electron density corresponding to the 191-199 loop
is less well defined even though the NAD+ site appears to
be fully occupied. This implies that although the sulfate ion
establishes a series of interactions with both the protein and the
cofactor, these are less efficient in stabilizing the 191-199 loop
than those formed by the covalently bound 2'-phosphate. Once
stabilized, the 191-199 loop appears to protect the coenzyme from
solvent as the NADP+ accessible surface (42) is reduced
from 122 Å2 when the loop is removed to 38 Å2
when it is well ordered.
The E2 molecule and the residues forming the steroid
binding site are well superposed in the two wild-type structures
(Table III). On the other hand, the rms
differences values resulting from the superposition of each mutant onto
the wild-type models show a significant deviation for estradiol,
relative to the residues involved in the hydrophobic site
(Fig. 6). In this respect, the loss of a
hydrogen bond between residue 221 and estradiol may be responsible for
the increased steroid mobility observed in the H221L mutant. There is
also a significant difference in substrate position between two
non-equivalent monomers such as mC and mD. The slight substrate
reorientation observed in the H221Q mutant is likely to be due to a
shift of the hydrogen bond between E2-O3 and
Gln221-N
1.
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Table III
Estradiol positional differences
Superpositions were made with the ALIGN program (52) on the substrate
binding site residues (Ser142, Val143, Met147,
Leu149, Try155, Pro187, Try218,
Val225, Phe226, Phe259, and Met279).
Monomer mC of the H221L · E2 · NADP+
complex was used for the superposition with the wild-type enzyme. 1fds
and 1fdt are Protein Data Bank accession codes for 17 -HSD1 · E2 and 17 -HSD1 · E2 · NADP+ complexes,
respectively.
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Fig. 6.
Stereoscopic view of the steroid binding site
of wild-type (1fdt model in yellow, and 1fds model in
orange), H221L (monomers mC in blue, mD in
light blue), and H221Q (pink).
Phe226 of the 1fdt model and Gln221 of the
H221Q mutant are shown with their two modeled conformations. The
steroid environment is very well conserved, especially
Phe155 and Ser142 which are the residues
involved in the catalytic reaction. Significant deviations are observed
for the steroid, especially for its C and D rings.
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The catalytic efficiency is strongly affected by mutations at the
His221 position (Table IV).
Reduction is decreased 10-fold for the H221L mutant and 4-fold for the
H221Q mutant (18-fold and 6-fold, respectively, for the oxidation).
This may be partially explained by a 4.4-fold increase in the reduction
reaction Km for H221L and a respective 2.3-fold
increase for the H221Q mutant (4-fold and 3.4-fold, respectively, for
the oxidation reaction). As the histidine-to-glutamine mutation
preserves the hydrogen bond with C3-OH, this interaction
seems to be essential for the catalytic activity. As expected from the
structure, the H221L mutation has a limited effect on the cofactor
binding site architecture, the Km value for
NADP+ reduction being close to that of the wild-type enzyme
(Table V).
 |
DISCUSSION |
The Role of the 191-199 Loop--
The H221L mutant provides the
first image of a well ordered 191-199 loop in a 17
-HSD1 structure.
The conformation of this loop in the
H221L·E2·NADP+ complex is very likely
to be identical to that of the wild-type enzyme when fully complexed to
the cofactor. This is supported, on the one hand, by biochemical
results that indicate that the affinity of the H221L mutant enzyme for
NADP+ is similar to that of the wild-type protein (Table V)
and, on the other, by the fact that the 191-199 loop is located at the protein surface, and it is not involved in potentially constraining crystal packing interactions in either structure. The stabilization of
the 191-199 loop, that is disordered in the absence of cofactor, is
mediated by the interactions of Lys195 and
Phe192 with NADP+. Furthermore, loop residues
Phe192 and Met193 shield NADP+ from
solvent and contribute to the hydrophobic character of the nicotinamide
binding pocket.
Other dehydrogenases also have a similarly flexible loop near their
dinucleotide binding site. One example is the mobile loop comprising
residues 16-20 in Escherichia coli dihydrofolate reductase which is involved in hydride transfer (43). This loop, which becomes
well ordered only in the enzyme·NADP+·folate
complex, has been found to shield the nicotinamide moiety from solvent
and to participate on the transition-state stabilization (44).
Replacement of the Met16-Ala19 stretch by
glycine results in a 550-fold decrease in the hydride transfer rate.
Another case is lactate dehydrogenase. As in 17
-HSD1, a loop
comprising residues 97-123 is stabilized by interactions with NADH and
shields the cofactor from solvent (45). In the SDR family, E. coli 7
-HSD (23) exhibits a large conformational change of the
195-210 loop upon substrate binding that also results in shielding of
the catalytic site. In the 3
,20
-HSD structure (19), a small
conformational change of the 184-189 loop located close to the active
site is also observed. In the MLCR·NADP+ complex (22),
the Met186 interaction with the nicotinamide moiety is
similar to the one observed between Phe192 and the
NADP+ in 17
-HSD1 (Fig. 3). However, it is not known
whether the 184-190 loop has the same conformation in the MLCR
apoenzyme.
All the SDR loops described above are located between the
F sheet
and the
G helix and are flanked by two proline residues (Pro187 and Pro200 in 17
-HSD1). As it was
previously suggested by Tanaka et al. (23), these two
prolines may prevent conformational changes from propagating to the
rest of the protein. An amino acid sequence alignment of SDR enzymes
reveals that a proline residue located at position 185 (17
-HSD1
numbering) is highly conserved, except for 17
-HSD1, where this
residue is found at position 187 (Fig. 7). Furthermore, a hydrophobic residue,
equivalent to Phe192 of 17
-HSD1, is found in 38 out of
the 46 amino acid sequences analyzed (Fig. 7). These loops, that are
stabilized by both substrate or cofactor, may be characteristic of
enzymes belonging to the SDR family. In turn, their protection of the
nicotinamide moiety and contribution of hydrophobic residues to the
active site structure suggest they are involved in catalytic hydride
transfer.

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Fig. 7.
Sequence alignment of the known
NADP+-preferring SDR enzymes in the 1-50 and 185-200
(17 -HSD1 numbering) regions. Homology rates above 60% (based
on Risler table (51)) are depicted in red. Residues
Ser11, Arg37, Phe192, and
Lys195 are labeled with a red triangle,
Gly9, Gly13, and Gly15 are labeled
with a cyan circle, and Pro187 is labeled with a
green star. The alignment was obtained with CLUSTALW and
corrected with the help of a structural alignment of all the known
SDR-structures. Shown are: DHB1_HUMAN, human type 1 17 -hydroxysteroid dehydrogenase; DHB1_MOUSE, mouse type 1 17 -hydroxysteroid dehydrogenase; DHB1_RAT, rat type 1 17 -hydroxysteroid dehydrogenase; AP27_MOUSE, mouse lung carbonyl
reductase; DHB3_HUMAN, human type 3 17 -hydroxysteroid dehydrogenase;
BA71_EUBSP, Eubacterium 7 -hydroxysteroid dehydrogenase; DHCA_HUMAN,
human 15 hydroxyprostaglandin dehydrogenase; DHCA_MOUSE, mouse 15 hydroxyprostaglandin dehydrogenase; DHCA_RABBIT, rabbit 15 hydroxyprostaglandin dehydrogenase; DHCA_RAT, rat 15 hydroxyprostaglandin dehydrogenase; DHG1_BACME, Bacillus
megaterium glucose-1-dehydrogenase I; DHG2_BACME, Bacillus
megaterium glucose-1-dehydrogenase II; DHG3_BACME, Bacillus
megaterium glucose-1-dehydrogenase III; DHG4_BACME, Bacillus
megaterium glucose-1-dehydrogenase IV; DHGA_BACME, Bacillus
megaterium glucose-1-dehydrogenase-A; DHGB_BACME, Bacillus
megaterium glucose-1-dehydrogenase-B; DHG_BACME, Bacillus
megaterium glucose-1-dehydrogenase; DHG_BACSU, Bacillus
subtillis glucose-1-dehydrogenase; DHI1_HUMAN, human
corticosteroid 11 -dehydrogenase; DHI1_MOUSE, mouse corticosteroid
11 -dehydrogenase; DHI1_RAT, rat corticosteroid 11 -dehydrogenase;
DHI1_SHEEP, sheep corticosteroid 11 -dehydrogenase; DLTE_BACSU,
Bacillus subtillis DLTE protein; FABG_ARATH,
Arabidopsis thaliana 3-oxoacyl-[acyl-carrier protein]
reductase; FABG_BRANA, Brassica napus
3-oxoacyl-[acyl-carrier protein] reductase; FABG_CULPA, Cuphea
lanceolata 3-oxoacyl-[acyl-carrier protein] reductase;
FABG_ECOLI, Escherichia coli 3-oxoacyl-[acyl-carrier
protein] reductase; FABG_HAEIN, Hemophilus influenzae
3-oxoacyl-[acyl-carrier protein] reductase; FABG_VIBHA, Vibrio
harveyi 3-oxoacyl-[acyl-carrier protein] reductase; GNO_GLUOX,
Gluconobacter oxydans gluconate 5-dehydrogenase; HDHA_CLOSO,
Clostridium sordellii 7 - hydroxysteroid dehydrogenase;
PHAB_ACISP, Acinetobacter sp. acetoacyl_CoA reductase;
PHAB_PARDE, Paracoccus denitrificans acetoacyl_CoA
reductase; PHBB_ALCEU, Alcaligenes eutrophus acetoacyl_CoA
reductase; PHBB_CHRVI, Chromatium vinosum acetoacyl_CoA
reductase; PHBB_RHIME, Rhizobium meliloti acetoacyl_CoA
reductase; PHBB_ZOORA, Zoogloea ramigera acetoacyl_CoA
reductase; PTR1_LEIMA, Leishmania major pteridine 1 reductase; PTR1_LEITA, Leishmania tarentolae pteridine 1 reductase; ROH2_RAT, rat type 2 retinol dehydrogenase; ROH3_RAT, rat
type 3 retinol dehydrogenase; TRN1_DATST, Datura stramonium
tropinone reductase-I; TRN2_DATST, Datura stramonium
tropinone reductase-II; TRN2_HYONI, Hyoscyamus niger
tropinone reductase-II; TRNH_DATST, Datura stramonium
tropinone reductase; and YCIK_ECOLI, Escherichia coli
hypothetical oxidoreductase in btur-sohb intergenic region.
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The Coenzyme Specificity--
As indicated by the different x-ray
complex structures, and by previous biochemical results (7), 17
-HSD1
is able to bind both NADP+ and NAD+ cofactors.
The 17
-HSD1·NADP+ complex has been shown to be
stabilized through hydrogen bonds established between the dinucleotide
2'-phosphate moiety and the main chain NHs of residues
Cys10, Ser11, and Arg37 (25). In
most known structures of NADP+-preferring enzymes, the two
negative charges of the 2'-phosphate group are compensated by one or
two positively charged residues. Due to disorder of the 191-199 loop,
these interactions were not observed in our NADP+/wild-type
17
-HSD1 complex structure.
Now, the H221L mutant structure with its well defined 191-199 loop,
allows us to definitely classify 17
-HSD1 among the
NADP+-preferring enzymes. Three residues,
Ser11, Arg37, and Lys195, interact
with the dinucleotide 2'-phosphate group of the cofactor (Fig. 5).
Arg37 forms a salt bridge that compensates one of the two
negative charges of the phosphate. Incidentally, this residue, as
Arg39, is also present in the MLCR·NADP+
complex (22). In both structures, it is located in the turn between the
-sheet B and the
-helix C of the
A-
B-
B-
C-
C-
D-
D motif that forms the Rossmann fold
(Figs. 1 and 7). This arginine is conserved in 26 of the 46 proteins
known to be NADP+-preferring enzymes (46) (Fig. 7).
A second basic residue, located in the 4th position of the
Gly-Xaa-Xaa-Xaa-Gly-Xaa-Gly consensus sequence of the dinucleotide binding motif, often further compensates the 2'-phosphate charge. Indeed, sequence alignment studies reveal that this residue is conserved in 54% of the NADP+-preferring SDR proteins
(Fig. 7). In 17
-HSD1, there is no basic residue in the 4th position.
Instead, the 2'-phosphate group interacts with Ser11, which
is located in the third position of the consensus sequence. In fact,
among the 21 NADP+-preferring enzymes of the SDR family
that do not have a basic residue at this position, 15 are found to have
either serine or threonine instead (Fig. 7). In 17
-HSD1, further
charge compensation of the 2'-phosphate group is afforded by
Lys195. This residue is not conserved in the SDR family of
amino acid sequences, and superposition of the known SDR structures
shows that it is located in a variable loop. Moreover, in most of the NADP+·enzyme complex structures having a Rossmann-fold
binding motif (47), the 2'-phosphate group interacts with one to three
residues located in either the
A
B or the
B
C turn (Fig. 1).
Thus 17
-HSD1 is the first SDR structure for which such an atypical
charge compensation is observed. This may be relevant to the
understanding of the evolution of the NADP(H) binding motif.
The Role of His221 in Substrate Specificity--
In
the wild-type structure, His221 was found to hydrogen bond
to the C3-OH of the estradiol moiety. This hydrogen bond is
preserved in the H221Q mutant. In the H221L mutant, however, the
mutated leucine residue can only participate in the formation of the
hydrophobic pocket of the steroid binding site. Consequently, it can be
concluded that this hydrogen bond is not essential to substrate
binding. However, comparison of all the available x-ray structures
shows that, as a function of the immediate environment, estradiol can occupy slightly different positions in its hydrophobic pocket and that
the largest differences concern its C and D rings (Fig. 6). The lack of
hydrogen bond formation in the H221L mutant reduces the catalytic
efficiency by 9 to 18-fold. This can be compared with a 4- to 5-fold
reduction in the H221Q mutant (Table IV). Taken together, these results
suggest that the hydrogen bond between His221 and the
C3-OH group is important for enzyme specificity because it
helps in establishing the catalytically relevant steroid/protein
interactions of O17, Tyr155 O
,
and Ser142 O
. These observations may explain
the specificity of 17
-HSD1 for aromatic A ring-containing
substrates. With substrates like testosterone or 4-androstenedione, the
orientation of the C3-O3 bond relative to the
C17-O17 bond is different. Accordingly, we
suggest that for these substrates, the interaction of
His221 with the O3 atom would place the
O17 in a position which is unfavorable for catalysis.
We thank J.-L. Ferrer and M. Roth (IBS,
Grenoble) for help using the D2AM ESRF beam line.