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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 enzyme’s preference for NADP(H) more than 20-fold. Substitution of the negatively charged residue, aspartic acid, into the Leu36 position switched the enzyme’s 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 enzyme’s 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 enzyme’s 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}-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{gamma} 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{alpha}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 {alpha}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 enzyme’s NADP(H) preference, whereas Leu36Asp changed the enzyme’s 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go, 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. 1CGo 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.



View larger version (30K):
[in this window]
[in a new window]
 
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 2–7, 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).

 
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 1Go. 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 number–the 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 1Go) 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 enzyme’s 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. 2Go and 3Go). The variation with this positively charged residue, therefore, only enhanced the enzyme’s reductase activity in cells.


View this table:
[in this window]
[in a new window]
 
Table 1. Kinetic Parameters of Human 17ß-HSD 1 Wild Type and Variants Ser12Lys and Leu36Asp

 


View larger version (24K):
[in this window]
[in a new window]
 
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.

 


View larger version (16K):
[in this window]
[in a new window]
 
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.

 
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. 2Go and 3Go). This variation thus altered the enzyme’s 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. 4Go). 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 2Go and Fig. 5Go). The discrepancies in activity between purified enzyme and intact cells may be caused by instabilities in the variant after cell disruption.



View larger version (29K):
[in this window]
[in a new window]
 
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 ).

 

View this table:
[in this window]
[in a new window]
 
Table 2. Kinetic Parameters of 17ß-HSD1 Wild Type and the Ser142Gly, His221Ala, and Glu282Ala Variants

 


View larger version (18K):
[in this window]
[in a new window]
 
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.

 
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 2Go). The kinetic constants changed more significantly during the estrone reduction using NADPH as the cofactor (Table 2Go), but in intact cells, the estrone reduction activity was not significantly altered (Fig. 5Go) (for explanation, see Discussion). However, the variant Glu282Ala did not show any significant modification either in vitro or in intact Sf9 cells.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 6Go). 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. 6BGo). 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 enzyme’s NADP(H) preference while keeping the enzyme’s 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 enzyme’s NADP(H) preference but does not interfere with the enzyme’s 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.



View larger version (14K):
[in this window]
[in a new window]
 
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.

 
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 enzyme’s 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 manufacturer’s instructions. Table 3Go 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].


View this table:
[in this window]
[in a new window]
 
Table 3. Oligonucleotide Primers Used for Site-Directed Mutagenesis

 
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 manufacturer’s 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 4–5 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 (1.8–2 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 20–40 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 d’Urfé, 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 70–90 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 0.007–5 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 0.014–25 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 0.1–5000 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-E2–17ß-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 l’Aide à la Recherche/Fonds de la Recherche en Santé du Québec.

1 These authors made the same contribution for variant construction. Back

2 Current address: Agriculture et Agroalimentaire Canada, Station de Recherchers sur les Sols et les Grandes Cultures, Sainte-Foy, Québec, Canada. Back

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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Tobias B, Covey DF, Strickler RC 1982 Inactivation of human placental 17ß-estradiol dehydrogenase and 20{alpha}-hydroxysteroid dehydrogenase with active site-directed 17ß-propenyl-substituted progestin analogs. J Biol Chem 257:2783–2786[Free Full Text]
  2. Luu-The V, Zhang Y, Poirier D, Labrie F 1995 Characteristics of human type 1, 2 and 3 17ß-hydroxysteroid dehydrogenase activities: oxidation/reduction and inhibition. J Steroid Biochem Mol Biol 55:581–587[CrossRef][Medline]
  3. Poulin R, Labrie F 1986 Stimulation of cell proliferation and estrogenic response by adrenal C19-{delta} 5-steroids in the ZR-75–1 human breast cancer cell line. Cancer Res 46:4933–4937[Abstract]
  4. Aakvaag A, Utaaker E, Thorsen T, Lea OA, Lahooti H 1990 Growth control of human mammary cancer cells (MCF-7 cells) in culture: effect of estradiol and growth factors in serum-containing medium. Cancer Res 50:7806–7810[Abstract]
  5. Yue W, Wang JP, Hamilton CJ, Demers LM, Santen RJ 1998 In situ aromatization enhances breast tumor estradiol levels and cellular proliferation. Cancer Res 58:927–932[Abstract]
  6. Labrie F 1991 At the cutting edge: intracrinology. Mol Cell Endocrinol 78:c113–c118
  7. Bulun SE, Price TM, Aitden J, Mahendroos MS, Simpson ER 1993 A link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription. J Clin Endocrinol Metab 77:1622–1628[Abstract]
  8. Suzuki T, Moriya T, Ariga N, Kaneko C, Kanazawa M, Sasano H 2000 17ß-Hydroxysteroid dehydrogenase type 1 and type 2 in human breast carcinoma: a correlation to clinicopathological parameters. Br J Cancer 82:518–523[CrossRef][Medline]
  9. Sasano H, Suzuki T, Takeyana J, Utsunomiya H, Ito K, Ariga N, Moriya T 2000 17-ß-Hydroxysteroid dehydrogenase in human breast and endometrial carcinoma: a new development in intracrinology. Oncology 59:5–12[CrossRef][Medline]
  10. Reed MJ 1991 Oestradiol-17ß hydroxysteroid dehydrogenase: its family and function. J Endocrinol 129:163–165[Medline]
  11. Luu-The V, Labrie C, Zhao HF, Couët J, Lachance Y, Simard J, Leblanc G, Labrie F 1989 Characterization of cDNAs for human estradiol 17ß-dehydrogenase and assignment of the gene to chromosome 17: evidence of two mRNA species with distinct 5'-termini in human placenta. Mol Endocrinol 3:1301–1309[Abstract]
  12. Lin SX, Yang F, Jin JZ, Breton R, Zhu DW, Luu-The V, Labrie F 1992 Subunit identity of the dimeric 17ß-hydroxysteroid dehydrogenase from human placenta. J Biol Chem 267:16182–16187[Abstract/Free Full Text]
  13. Ryan KJ, Engel LL 1953 The interconversion of estrone and estradiol by human tissue slices. Endocrinology 52:287–291[Medline]
  14. Martel C, Rheaume E, Tanahashi M, Trudel C, Couët J, Luu-The V, Simard J, Labrie F 1992 Distribution of 17ß-hydroxysteroid dehydrogenase gene expression and activity in rat and human tissues. J Steroid Biochem Mol Biol 41:597–603[CrossRef][Medline]
  15. Persson B, Krook M, Jörnvall H 1991 Characteristics of short-chain alcohol dehydrogenases and related enzymes. Eur J Biochem 200:537–543[Abstract]
  16. Ghosh D, Pletnev VZ, Zhu DW, Wawrzak Z, Duax WL, Pangborn W, Labrie F, Lin SX 1995 Structure of human estrogenic 17ß-hydroxysteroid dehydrogenase at 2.20 Å resolution. Structure 3:503–513[Medline]
  17. Krozowski Z 1992 11ß-Hydroxysteroid dehydrogenase and the short-chain alcohol dehydrogenase (SCAD) superfamily. Mol Cell Endocrinol 84:C25–C31
  18. Jin JZ, Lin SX 1999 Human estrogenic 17ß-hydroxysteroid dehydrogenase: predominance of estrone reduction and its induction by NADPH. Biochem Biophys Res Commun 259:489–493[CrossRef][Medline]
  19. Azzi A, Rhese PH, Zhu DW, Campbell RL, Labrie F, Lin SX 1996 Crystal structure of human estrogenic 17ß-hydroxysteroid dehydrogenase complexed with 17ßestradiol. Nat Struct Biol 3:665–668[Medline]
  20. Breton R, Housset D, Mazza C, Fontecilla-Camps JC 1996 The structure of a complex of human 17ß-hydroxysteroid dehydrogenase with estradiol and NADP+ identifies two principal targets for the design of inhibitors. Structure 4:905–915[Medline]
  21. Sawicki MW, Erman M, Puranen T, Vihko P, Ghosh D 1999 Structure of the ternary complex of human 17ß-hydroxysteroid dehydrogenase type 1 with 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin) and NADP+. Proc Natl Acad Sci USA 96:840–845[Abstract/Free Full Text]
  22. Tanaka N, Nonaka T, Nakanishi M, Deyashiki Y, Hara A, Mitsui Y 1996 Crystal structure of the ternary complex of mouse lung carbonyl reductase at 1.8 Å resolution: the structural origin of coenzyme specificity in the short-chain dehydrogenase/reductase family. Structure 4:33–45[Medline]
  23. Nakanishi M, Kakumoto M, Matsuura K, Deyashiki Y, Tanaka N, Nonaka T, Mitsui Y, Hara A 1996 Involvement of two basic residues (Lys-17 and Arg-39) of mouse lung carbonyl reductase in NADP(H)-binding and fatty acid activation: site-directed mutagenesis and kinetic analyses. J Biochem 120:257–263[Abstract]
  24. Nakanishi M, Matsuura K, Kaibe H, Tanaka N, Nonaka T, Mitsui Y, Hara A 1997 Switch of coenzyme specificity of mouse lung carbonyl reductase by substitution of threonine 38 with aspartic acid. J Biol Chem 272:2218–2222[Abstract/Free Full Text]
  25. Grimshaw CE, Matthews DA, Varughese KI, Skinner M, Xuong NH, Bray T, Hoch J, Whiteley JM 1992 Characterization and nucleotide binding properties of a mutant dihydropteridine reductase containing an aspartate 37-isoleucine replacement. J Biol Chem 267:15334–15339[Abstract/Free Full Text]
  26. Chen Z, Lee W, Chang SH 1991 Role of aspartic acid 38 in the cofactor specificity of Drosophila alcohol dehydrogenase. Eur J Biochem 202:263–267[Abstract]
  27. Chen Z, Jiang J, Lin ZG, Lee WR, Baker ME, Chang SH 1993 Site-specific mutagenesis of Drosophila alcohol dehydrogenase: evidence for involvement of tyrosine-152 and lysine-156 in catalysis. Biochemistry 32:3342–3346[Medline]
  28. Puranen TJ, Poutanen MH, Peltoketo HE, Vihko PT, Vihko RK 1994 Site-directed mutagenesis of the putative active site of human 17ß-hydroxysteroid dehydrogenase type 1. Biochem J 304:289–293[Medline]
  29. Puranen T, Poutanen M, Ghosh D, Vihko P, Vihko R 1997 Characterization of structural and functional properties of human 17ß-hydroxysteroid dehydrogenase type 1 using recombinant enzymes and site-directed mutagenesis. Mol Endocrinol 11:77–86[Abstract/Free Full Text]
  30. Huang YW, Lu ML, Qi H, Lin SX 2000 Membrane-bound human 3ß-hydroxysteroid dehydrogenase: overexpression with his-tag using a baculovirus system and single-step purification. Protein Expr Purif 18:169–174[CrossRef][Medline]
  31. Alnemri ES, Fernandes-Alnemri T, Nelki DS, Dudly K, Dubois GC, Litwack G 1993 Overexpression, characterization, and purification of a recombinant mouse immunophilin FKBP-52 and identification of an associated phosphoprotein. Proc Natl Acad Sci USA 90:6839–6843[Abstract]
  32. Gaillard I, Slotboom DJ, Knol J, Lolkema JS, Konings WN 1996 Purification and reconstitution of the glutamate carrier GltT of thermophilic bacterium Bacillus stearothermophilus. Biochemistry 35:6150–6156[CrossRef][Medline]
  33. Loo TW, Clarke DM 1995 Rapid purification of human P-glycoprotein mutants expressed transiently in HEK 293 cells by nickel-chelated chromatography and characterization of their drug-stimulated ATPase activity. J Biol Chem 270:21449–21452[Abstract/Free Full Text]
  34. David NE, Gee M, Andersen B, Naider F, Thorner J, Stevens RC 1997 Expression and purification of the Saccharomyces cerevisiae {alpha}-factor receptor (Step2p), a 7-transmembrane-segment G protein-coupled receptor. J Biol Chem 272:15553–15561[Abstract/Free Full Text]
  35. Stryer L 1988 Biochemistry. New York: W.H. Freeman and Co.; 430–431
  36. Sem DS, Kasper CB 1993 Interaction with arginine 597 of NADPH-cytochrome P-450 oxidoreductase is a primary source of the uniform binding energy used to discriminate between NADPH and NADH. Biochemistry 32:11548–11558[Medline]
  37. Kubiseski TJ, Flynn TG 1995 Study on human aldose reductase. J Biol Chem 270:16911–16917[Abstract/Free Full Text]
  38. Mazza C, Breton R, Housset D, Fontecilla-Camps JC 1998 Unusual charge stabilization of NADP+ in 17ß-hydroxysteroid dehydrogenase. J Biol Chem 273:8145–8152[Abstract/Free Full Text]
  39. Labrie F, Luu-The V, Lin SX, Labrie C, Simard J, Breton R, Belanger A 1997 The key role of 17ß-hydroxysteroid dehydrogenases in sex steroid biology. Steroids 62:148–158[CrossRef][Medline]
  40. Oppermann UCT, Filling C, Berndt KD, Persson B, Benach J, Ladenstein R, Jornvall H 1997 Active site directed mutagenesis of 3ß/17ß-hydroxysteroid dehydrogenase establishes differential effects on short-chain dehydrogenase/reductase reactions. Biochemistry 36:34–40[CrossRef][Medline]
  41. Gangloff A, Garneau A, Huang YW, Yang F, Lin SX 2001 Human estrogenic 17ß-hydroxysteroid dehydrogenase specificity: enzyme regulation through an NADPH-dependent substrate inhibition towards the highly specific estrone reduction. Biochem J 356:269–276[CrossRef][Medline]
  42. Eszes CM, Sessions RB, Clarke AR, Moreton KM, Holbrook JJ 1996 Removal of substrate inhibition in a lactate dehydrogenase from human muscle by a single residue change. FEBS Lett 399:193–197[CrossRef][Medline]
  43. Higuchi RK, Krummel B, Saiki RK 1988 A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 16:7351–7367[Abstract]
  44. Luckow VA, Lee SC, Barry GF, Olins PO 1993 Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol 67:4566–4579[Abstract]
  45. Breton R, Yang F, Jin JZ, Li B, Labrie F, Lin SX 1994 Human 17ß-hydroxysteroid dehydrogenase: overproduction using a baculovirus expression system and characterization. J Steroid Biochem Mol Biol 50:275–282[CrossRef][Medline]
  46. Guex N, Peitsch MC 1997 SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723[Medline]
  47. Gunsteren WF, Billeter SR, Eising AA, Hünenberger PH, Krüger P, Mark AE, Scott WRP, Tironi IG 1996 Biomolecular simulation: the GROMOS 96 manual and user guide. Zürich, Switzerland: Hochschulverlag AG