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Biocenter Oulu and Department of Clinical Chemistry (T.P., M.P., R.V., P.V.), University of Oulu, Oulu, Finland; the Hauptman-Woodward Medical Research Institute, Inc. (D.G.), Buffalo, New York 14203; and Roswell Park Cancer Institute (D.G.), Buffalo, New York 14263
Address all correspondence and requests for reprints to: Prof. Pirkko Vihko, Biocenter Oulu and Department of Clinical Chemistry, University of Oulu, Kajaanintie 50, FIN-90220 Oulu, Finland. E-mail: pvihko{at}whoccr.oulu.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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So far, five distinct 17-HSD enzymes have been described. Four of
the five (types 1–4) (3, 10, 11, 12, 13) belong to the short-chain
dehydrogenase/reductase (SDR) protein family (14), whereas 17-HSD type
5 is a member of the aldoketoreductase family (15, 16). The enzymes of
the SDR family contain two highly conserved regions, namely a ß
ß
Rossman fold at the amino terminal end of the enzymes (17, 18) and a
conserved Tyr-X-X-X-Lys (X is any amino acid) motif (19). Several
studies indicate that these conserved structural elements are critical
for cofactor-binding and hydride transfer, respectively (20, 21, 22, 23, 24, 25, 26, 27, 28). These
facts, together with the characterized x-ray structures of the family
members (29, 30, 31, 32, 33, 34), suggest that enzymes in the SDR family have
structural similarities (14) and are functionally related. However, the
overall identity between 17-HSD enzymes is low. For instance, human
17-HSD types 1 and 3 share only about 20% sequence identity (3), and
yet both enzymes are able to catalyze the 17ß-reduction of estrone
and androstenedione (3, 35, 36). The human type 1 enzyme prefers
estrone as a substrate, whereas the type 3 enzyme prefers
androstenedione. The structural motifs and amino acids related to the
substrate specificity of the steroid-metabolizing enzymes in the SDR
family are, however, only superficially known.
Interestingly, our recent studies have indicated that although human and mouse 17-HSD type 1 enzymes have 63% amino acid identity, the mouse enzyme uses both estrone and androstenedione equally as a substrate in cultured cells (37). However, the data show that similar to the human enzyme, rodent 17-HSD type 1 is involved in glandular estradiol biosynthesis (37, 38, 39). The present study was carried out to clarify the structural elements resulting in differential substrate specificity of human and rodent 17-HSD type 1 enzymes, using rat/human 17-HSD type 1 chimeric proteins as well as site-directed substitutions in the enzymes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of rat/human chimeric and substituted 17-HSD type 1
enzymes
Six rat/human chimeric 17-HSD type 1 enzymes were generated
using the PCR technique and/or restriction enzyme sites present in
the human and rat 17-HSD type 1 cDNAs (Fig. 1A
).
When PCR was used to construct the aminoterminal fragment of rat enzyme
or the carboxyterminal fragment of the human enzyme, the flanking
primers, 5'-TTATATTAGCGGCCGCGGTAGGACCCTTCATGCT-3' and
5'-TATATGAATTCAGGAAGCCTTTACTGCGGGGC-3' were used, respectively.
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). To generate the C-terminal fragment (amino acids 122–327), human
17-HSD type 1 cDNA was digested with PstI and
EcoRI enzymes. Thereafter, the isolated fragment was ligated
to the PCR-amplified N-terminal part of the rat enzyme.
R148/H149, R170/H171, and R184/H185 consisted of 148, 170, and 184
N-terminal amino acids of rat type 1 enzyme, respectively, associated
with C-terminal residues of the human enzyme, corresponding to amino
acids of 149–327, 171–327, and 185–327. Aminoterminal fragments of
these chimeric enzymes were amplified by PCR using intact rat 17-HSD
type 1 cDNA as a template. Similarly, full length human 17-HSD type 1
cDNA was used as a template to amplify the carboxyterminal fragments
for the constructed rat/human chimeric proteins. Oligonucleotide
sequences designed for the PCR reactions are shown in Fig. 1A.
The R190/H191-chimer consisted of 190 aminoterminal amino acids of rat enzyme fused with 137 carboxyterminal amino acid (residues 191–327) of the human enzyme. To construct the protein, we utilized the fact that both the human and rat 17-HSD type 1 cDNAs contained restriction sites for ApaLI at identical positions. Both cDNAs were digested with EcoRI and ApaLI restriction enzymes, and the N-terminal fragment of the rat enzyme was ligated with the C-terminal fragment of human 17-HSD type 1.
The R266/H268 17-HSD type 1 enzyme consisted of aminoterminal residues
1–266 of rat enzyme fused with 60 carboxyterminal residues (amino
acids 268–327) of the human enzyme. To obtain the N-terminal part of
the chimeric protein, rat 17-HSD type 1 cDNA was digested with
EcoRI and FspI enzymes, and an
FspI-EcoRI-fragment containing human amino acids
268–327 was generated by PCR (Fig. 1A).
Nine amino acid substitutions
(Asn152HisAsp153Glu, Val171Ile,
Leu174ProPro175Leu,Leu180Val,Pro187Ala,
Asn152HisAsp153GluPro187Ala,Val171IlePro187Ala,
Leu174ProPro175LeuPro187Ala and
Leu180ValPro187Ala) in human 17-HSD type 1
were generated, replacing amino acids with those present in the rat
type 1 enzyme. The substitutions were generated using the
overlap-extension method (40), in a manner similar to that previously
described (27, 28). Substituted human 17-HSD type 1 cDNAs were
constructed using flanking primers
(5'-TTATATTAGCGGCCGCACCATGGCCCGCACCGTG-3' and
5'-TATATGAATTCAGGAAGCCTTTACTGCGGGGC-3') and the internal primers
presented in Fig. 1B. In PCR reactions, wild-type human 17-HSD type 1
cDNA was used as a template except in the cases of
Asn152HisAsp153GluPro187Ala,
Val171IlePro187Ala,
Leu174ProPro175LeuPro187Ala and
Leu180ValPro187Ala substitutions, in which
the constructed human Pro187Ala-substituted 17-HSD type 1
cDNA was used as a template. His152AsnGlu153Asp
substitution in rat 17-HSD type 1 was generated in a manner similar to
that described for human enzyme. The substituted rat cDNA-fragment was
amplified with flanking primers
(5'-TTATATTAGCGGCCGCGGTAGGACCCTTCATGCT-3' and
5'-TATATGAATTCTCTAGCTTAGATCTTTTGCTG-3') and the internal primers shown
in Fig. 1B, using intact rat 17-HSD type 1 cDNA as a template. Both
rat/human chimeric fragments and substituted 17-HSD type 1 cDNAs were
subcloned into Bluescript KS+ vectors (Stratagene, La
Jolla, CA). The constructs were sequenced to confirm that no unwanted
substitutions had occurred during amplification, and that ligations had
been accomplished properly.
Fragments coding for full-length human (10) and rat (38) 17-HSD type 1 cDNAs as well as the rat/human chimeric and substituted cDNAs were subcloned into pVL1392 nonfusion transfer vectors (Invitrogen). The constructs were cotransfected with modified baculovirus DNA into Sf9 cells, using the BaculoGold transfection system (Pharmingen, San Diego, CA). Recombinant proteins were produced in Sf9 insect cells in 600mL tissue-culture flasks at a cell density of 30 x 106 cells/flask. The cells were infected with recombinant 17-HSD type 1 Autographa californica nuclear-polyhedrosis viruses (17-HSD type 1-AcNPVs) at a multiplicity of infection of 1. The optimal level of expression was reached at about 70 h post infection, and the cells were then harvested by centrifugation at 1000 x g for 10 min.
Measurement of 17-HSD type 1 activity in vitro and in cultured
cells
The catalytic properties of the wild-type and substituted human
and rat 17-HSD type 1 enzymes were analyzed in intact cells in culture
and in cell homogenates in vitro. Activity measurements of
the rat/human chimeric 17-HSD type 1 enzymes were performed only in
cultured cells. Km and apparent Vmax values for
the human and rat 17-HSD type 1 enzymes were measured in
vitro as previously described (27, 28). For comparable results,
the same enzyme preparations were used to analyze both estrogenic and
androgenic activities. Briefly, the cells were homogenized in buffer A
(10 mM potassium phosphate buffer, pH 7.5, 1 mM
EDTA, 0.5 mM PMSF, 0.02% NaN3 and 20%
glycerol). For activity measurements, the 1000 x g
supernatants were diluted in 10 mM potassium phosphate, pH
7.5, containing 0.01% BSA, or alternatively in 10 mM
potassium phosphate, pH 7.5, containing 20% glycerol, 0.1%
N-octyl-ß-D-glucopyranoside and 0.01% BSA.
The enzyme preparations were then mixed with [3H]-labeled
E2, E1, A-dione, or T (0.73–7.3
µM substrate) and the reactions were started by adding a
cofactor (NAD+/NADH) to a final concentration of 1.3
mmol/liter. After a 20-sec incubation at 37 C, the reactions were
stopped, and the steroids were extracted into an organic phase (diethyl
ether:ethyl acetate 9:1). The substrates and products were then
separated using an acetonitrile/water (48:52, vol/vol) solution as a
mobile phase in a Symmetry C18 reverse-phase chromatography column
connected to an HPLC-system (Waters, Milton, MA). Km and
Vmax values for the 17-HSD type 1 enzymes were calculated
by using a GraFit-program (Erithacus Software Ltd., Staines, UK), which
fits data to the Michaelis Menten equation using nonlinear regression
analysis. The kinetic values represent the average ±SD of
at least three independent experiments. One micromole of product formed
per minute was defined as one unit of enzyme activity. Specific
activities (E1 
E2, A-dione T) for
substituted 17-HSD type 1 enzymes were measured similarly, using 37
µM substrate concentrations and 1 min incubations.
Activity measurements in cultured Sf9 insect cells were performed by
plating the cells into six-well plates at a density of 1.2 x
106 cells/well. The cells were allowed to attach for 1
h and were then infected with 17-HSD type 1-AcNPVs at a multiplicity of
infection of 2. At 50 h post infection, the reductive
(E1 E2 and A-dione T) and oxidative
(E2 E1 and T A-dione) 17-HSD activities
were measured in the intact cells in culture by adding 10
µM 3[H]-labeled substrate (20 nmol/well) to
the culture media. For comparable results, both estrogenic and
androgenic activities were measured after each infection. The media
were removed after 10, 20, 40, and 80 min of incubation, and substrate
and products were separated as described above. Measurements were
repeated at least three times, with identical results.
Student’s t test was used to analyze the differences between the mean values, which were regarded as statistically significant when the P values were below 0.01.
Modeling of rat 17-HSD type 1 protein
The three-dimensional structure of rat 17-HSD type 1 was built
by homology modeling using the crystal structure of the human enzyme
(31) and the amino acid sequence alignment as templates. All computer
calculations and graphics work were performed on a Silicon Graphics
Indigo Elan work station. Model building was carried out using the
software package CHAIN (41). The amino acid sequence of human 17-HSD
type 1 was substituted into that of rat enzyme. To accommodate a
deletion at position 229 in the rat enzyme (Ala229 in the
human enzyme), the C-terminus of the helix G' (31) was rebuilt. The
torsion angles of the side-chains were adjusted to avoid any
unfavorable contacts with other side-chains. The model was
energy-minimized by using the program XPLOR (42), with Engh and Huber
force fields (43). The energy minimization process converged after 180
cycles of positional refinement. Modeling of steroids in the active
site of 17-HSD type 1 enzymes was achieved by manually positioning and
adjusting a steroid molecule in the active site of the
three-dimensional structure and then energy-minimizing the complex. The
figures were created using SETOR software (44).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
In two sets of experiments performed in the present study, the relative
conversions of E1 to E2, and A-dione to T
(E1/A-dione ratio) were 4.68 ± 0.61 (SD)
and 5.33 ± 1.48 for the human enzyme, and 0.90 ± 0.12 and
0.85 ± 0.05 for the rat enzyme (Table 1, Fig. 3). Thus, in contrast to human 17-HSD type 1, the rat
enzyme was able to reduce androgens at least as efficiently as
estrogens in cultured cells. Both human and rat type 1 enzymes
catalyzed the opposite oxidative reactions far less efficiently (data
not shown).
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). The presence of the detergent was confirmed
not to affect the properties of the human enzyme (data not shown). In
line with that shown previously, the catalytic efficiency
(Vmax/Km) of human 17-HSD type 1 was
considerably greater (about 6-fold) for the reductive estrogenic
reaction (E1E2) compared with oxidation.
Similarly, the rat type 1 enzyme strongly preferred E1 over
E2 as a substrate (data not shown). In addition, the
Km values measured for the estrogenic activities were found
to be very similar for both human and rat enzymes (Fig. 2B). However,
in contrast to the estrogenic activity, significant differences between
the enzymes were detected in their ability to convert A-dione to T. The
rat enzyme catalyzed the reduction of A-dione with an efficiency (1.44)
very similar to that found for E1 (2.27), whereas the
catalytic efficiency of the human enzyme as regards estrogenic activity
was 715-fold higher than that of androgenic activity (Fig. 2B).
To evaluate the regions determining the difference between the
substrate specificities, rat/human chimeric enzymes were constructed,
and the substrate specificity of each enzyme was analyzed by comparing
the conversion of E1 to E2 and A-dione to T
(E1/A-dione ratio) in cultured Sf9 cells. The most striking
differences between the primary structures of human and rat enzymes are
located at the C-terminus (amino acids 285–327), and at the region
shown to be involved in the substrate binding of the enzymes (amino
acids 196–230). The amino acid identity between the proteins at these
regions is only 14% and 34%, respectively. Two chimeric enzymes were
constructed (R266/H268, R190/H191) to define the role of these regions
in substrate specificity (Fig. 1A). Interestingly, the R266/H268
chimeric enzyme possessed a substrate specificity very close to that
measured for the intact rat enzyme (Fig. 3), indicating that the
carboxy terminal part of 17-HSD type 1 is not involved in the
determination of steroid specificity of the enzyme. The chimeric enzyme
containing amino acids 191–327 of the human enzyme was significantly
(P < 0.001) more estrogen-specific
(E1/A-dione ratio 1.62, Fig. 3) than the intact rat enzyme.
However, the data indicated that the region from amino acid 190 through
268 accounted for only about one-third of the difference observed
between rat and human 17-HSD type 1 enzymes. Therefore, four additional
chimeric enzymes (R121/H122, R148/H149, R170/H171, R184/H185) were
constructed (Fig. 1A). The data demonstrated that both R121/H122 and
R148/H149 chimeric enzymes still retained substrate specificity
characteristic of the intact human enzyme (Fig. 3), indicating that the
first 149 amino terminal amino acids of 17-HSD type 1, including the
cofactor-binding region, are not critical for substrate specificity.
However, for the R170/H171 chimeric enzyme the E1/A-dione
ratio was significantly altered toward that found for wild-type rat
enzyme. With the R184/H185 chimeric enzyme the E1/A-dione
ratio was further decreased from that detected for the R170/H171
chimer, being closer to the catalytic properties of the R190/H191
chimer (Fig. 3). Thus, the regions mostly responsible for the
differences in the substrate specificity were located between amino
acids 148 and 268. These results are in line with the model of the
active site of rat 17-HSD type 1 (Fig. 4A), built on the
x-ray structure of the human enzyme (31). In contrast to that found for
human enzyme, the results indicated that an A-dione molecule is able to
bind to the active site of rat type 1 enzyme (Fig. 4A). Although the
precise structure of the steroid-binding cleft cannot be ascertained
from such three-dimensional homology modeling, the model revealed that
side chains of Leu149, His152,
Ala187, Tyr218, Tyr222,
Phe259, Met279, and Glu282 are in
the vicinity of the bound steroid, and that the side chains of residues
148–230 determine the androgenic activity of the rat enzyme.
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), suggesting that none of these
residues significantly contribute to the difference between human and
rodent enzymes. For
Asn152HisAsp153Glu-substituted human 17-HSD
type 1, the value for the E1/A-dione ratio was slightly
reduced as measured in cultured cells, but the substitutions resulted
in an enzyme with a lower estrogen specificity in vitro. The
data obtained in vitro are, thus, in line with those
obtained with the chimeric enzymes in cultured cells, and substituting
the corresponding amino acids of the rat enzyme with those present in
human 17-HSD type 1 (His152AsnGlu153Asp)
significantly increased the E1/A-dione ratio of the rat
enzyme both in vitro (P = 0.009) and in
cultured Sf9 cells (P < 0.001, Table 1). The
importance of Asn152HisAsp153Glu variation for
the substrate specificity in 17-HSD type 1 was further supported by
findings showing that in the crystal structure of human 17-HSD type 1
(Fig. 4B), the loop between residues Leu149 and
Asn152 is at a Van der Waals distance from the ß-face of
the A- and B-rings of estradiol, modeled at the active site. The
structural data also showed that these amino acids are vital to the
shape-complementarity of the steroid-binding cleft. Furthermore, the
side chain of Asn152 makes two hydrogen-binding contacts,
one to the main chain carbonyl oxygen of Pro150 and the
other to the Tyr218 side chain hydroxyl group (Fig. 4B).
Both of the interactions could be critical to the stability of the
conformation of the loop. Our modeling also showed that the methyl
group at the C10 position of the A-ring of A-dione could result in
steric hindrance against this loop at the active site. As a result of
the presence of His152 and Glu153 residues in
the region, the structure of the active site in the rat type 1 enzyme
is significantly altered at the proximity of the ß-face of the A- and
B-rings.
The difference in substrate specificities between R184/H185 and
R190/H191 chimeric enzymes, furthermore, indicated that the region
between amino acids 184–191 contains structures involved in substrate
specificity. Interestingly, the region possesses only one amino acid
difference: Pro187 in the human enzyme is replaced by Ala
in the rat enzyme. Substituting Ala in place of Pro in human 17-HSD
type 1 significantly (P = 0.002) decreased the
E1/A-dione ratio of the enzyme compared with the wild-type
human enzyme, as measured in cultured cells (Table 1). To find out
whether other amino acids together with Ala187 would
further affect steroid specificity, additional substitutions were
constructed in human 17-HSD type 1 in the region 148–191, namely
Asn152HisAsp153GluPro187Ala,Val171IlePro187Ala,
Leu174ProPro175LeuPro187Ala,
and Leu180ValPro187Ala. All the substitutions
had a mild effect on enzyme activity measured in vitro,
resulting in enzymes with lower E1/A-dione ratios compared
with Pro187Ala-substituted enzyme. However, in cultured
cells all the enzymes had a substrate specificity very similar to that
measured for the Pro187Ala-substituted enzyme. This
suggests that, of the amino acids in region 148–191, the
Asn152His, Asp153Glu and Pro187Ala
variations play the most significant role in determining the
differential substrate specificity of human and rat 17-HSD type 1. The
results, however, indicate that differential substrate specificity
between human and rat 17-HSD type 1 enzymes is achieved because of a
combination of several amino acid variations in the region between
residues 148–268, rather than one predominant change in the primary
structures of the enzymes. Structural elements found to be most
critical for the differential substrate specificities of human and rat
17-HSD type 1 enzymes are indicated in Fig. 5.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The carboxy termini of rat (38) and mouse (37) 17-HSD type 1 enzymes are 17 amino acids longer than that of the human enzyme (10). In addition, the C-terminal regions of the rodent enzymes are more hydrophobic than that of the human enzyme. In this study, rat 17-HSD type 1 was found to be rapidly inactivated in vitro without a nonionic detergent, N-octyl-ß-D-glucopyranoside. Thus, it is possible that for proper folding and to maintain full enzymatic activity, rodent enzymes might require membrane contact via the hydrophobic C-terminal region, which is not present in the human enzyme. Structural data reveal (31) that residues beyond position 230 are not directly involved in building the steroid-binding cleft. The finding in the present study indicating that the R266/H268 chimeric enzyme has a substrate specificity similar to that of the rat enzyme is consistent with the structural data. However, the C-terminal end of 17-HSD type 1 could be essential in stabilization of the enzyme.
Although the overall identity between human and rodent 17-HSD type 1 is
63%, the identity between the first 200 amino acids is 81%. The
ßA-B-ßB-C-ßC-D-ßD-fold present in the first 100 amino
terminal residues of the enzymes is involved in the folding of the
cofactor-binding region of the enzyme (31). Residues from 100 to 150
comprise the helix E and the strand ßE, which are fully conserved
between the human and rodent enzymes. The E helix has been shown to
be involved in dimerization of the enzyme monomers (28, 31).
Altogether, modeling studies showed that the three-dimensional
structure of rat 17-HSD type 1 in the cofactor-binding region and
beyond, up to about Pro150, is nearly identical to that of
the human enzyme. The present finding that R121/H122 and R148/149
chimeric enzymes showed virtually no change in their substrate
specificities in comparison with the human enzyme, is in complete
agreement with the structural data.
The present data with chimeric enzymes showed that the region formed by
residues 148–266 determines most of the steroid specificity of the
17-HSD type 1 enzyme. The chimeric enzymes constructed, namely
R170/H171, R184/H185, R190/H191 and R266/H268, demonstrated a
consistent decrease in the E1/A-dione ratio along with more
C-terminal residues from the rat enzyme. This is in line with the
structural data because the recognition end of the active site is
primarily at residues 185 to 230, which is also the region with the
greatest number of amino acid differences between the human and rat
enzymes (31), including 23 amino acid variations and one deletion
(Ala229 in human enzyme). Modeling of the structure of the
rat enzyme suggests that the shape of the active site at this end would
be altered as a result of a number of substitutions on the surface of
the helix facing the active site cleft and a deletion at the C-terminus
of G'.
A major part of the difference between human and rat enzymes was localized at the region between amino acids 148–191. At this region, there are only a few amino acid differences between the enzymes, of which Asn152His, Asp153Glu and Pro187Ala variations were found to be most closely related to steroid specificity. The modeling studies revealed that an His residue at position 152 could remarkably alter the hydrogen-bond formation pattern associated with the Asn residue, and hence the conformation of the loop which is located in the vicinity of the A- and B-rings of the bound substrate. Both of the changes could modify the steroid-binding pocket to one more favorable to androgenic substrates, and, hence, be partly responsible for the difference in substrate specificity between human and rodent enzymes.
A flexible region similar to that between Pro187 and
Pro200 in human 17-HSD type 1, defined by a short helical
turn, has recently been postulated to be important in
substrate-recognition and access to the active site in SDR family
members (46). Considerable movement of this region was observed in the
crystal structure of steroid-bound 7-HSD, and these two conserved
Pro residues were postulated to stabilize the rest of the main chain
(34). Pro187Ala substitution in human 17-HSD type 1 near
the A-ring of the steroid could, therefore, cause considerable
rearrangement of the active site, rendering it more like that of the
rat enzyme. To resolve the precise structural changes caused by
Pro187Ala substitution, the x-ray structures of rat enzyme
or one of the rat/human chimeric 17-HSD type 1 enzymes is, however,
required.
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Acknowledgments |
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Footnotes |
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Received March 10, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,20ß-hydroxysteroid dehydrogenase: a member of the short-chain
dehydrogenase family. Proc Natl Acad Sci USA 88:10064–10068,20ß-hydroxysteroid
dehydrogenase and possible roles of the residues conserved in
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-hydroxysteroid dehydrogenase from Escherichia
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