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Molecular Endocrinology, doi:10.1210/me.2006-0281
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Molecular Endocrinology 21 (2): 401-414
Copyright © 2007 by The Endocrine Society

Molecular Basis for the Aromatization Reaction and Exemestane-Mediated Irreversible Inhibition of Human Aromatase

Yanyan Hong, Bin Yu, Mark Sherman, Yate-Ching Yuan, Dujin Zhou and Shiuan Chen

Department of Surgical Research and Division of Information Sciences, Beckman Research Institute of the City of Hope, Duarte, California 91010

Address all correspondence and requests for reprints to: Shiuan Chen, Department of Surgical Research and Division of Information Sciences, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, California 91010. E-mail: schen{at}coh.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS AND DISCUSSION
 Conclusion
 MATERIALS AND METHODS
 REFERENCES
 
Aromatase converts androgens to aromatic estrogens. Aromatase inhibitors have been used as first-line drugs in the treatment of hormone-dependent breast cancer. Structural basis of the aromatization reaction and drug recognition by aromatase has remained elusive because of its unknown three-dimensional structure. In this study, recombinant human aromatase was expressed and purified from Escherichia coli. Using this purified and active preparation, the three-dimensional folding of aromatase was revealed by proteomic analysis. Combined with site-directed mutagenesis, several critical residues involved in enzyme catalysis and suicide inhibition by exemestane were evaluated. Based on our results, a new clamping mechanism of substrate/exemestane binding to the active site is proposed. These structure-function studies of aromatase would provide useful information to design more effective aromatase inhibitors for the prevention and the treatment of hormone-dependent breast cancer.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS AND DISCUSSION
 Conclusion
 MATERIALS AND METHODS
 REFERENCES
 
AROMATASE, A CYTOCHROME P450, catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogens. Upon receiving electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase, aromatase converts androstenedione and testosterone to estrone and estradiol, respectively. Estrogens are female hormones involved in the development and growth of breast tumors. Approximately 60% of premenopausal and 75% of postmenopausal breast cancer patients have estrogen-dependent carcinomas (1). Inhibition of aromatase is a new approach to treat estrogen-dependent breast cancer because the aromatization of androgen is the terminal and rate-limiting step in estrogen synthesis.

The third-generation aromatase inhibitors, developed in the early 1990s, including two nonsteroidal triazole derivatives [anastrozole (Arimidex, AstraZeneca Pharmaceuticals LP, Wilmington, DE) and letrozole (Femara, Novartis Pharmaceuticals Corp., East Hanover, NJ)] and one steroidal derivative [exemestane (Aromasin, Pharmacia & Upjohn S.p.A., Ascoli Picerno, Italy)] (Fig. 1Go), are widely used as the first-line drugs in the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients (2, 3, 4). These drugs were found to be better tolerated than tamoxifen and were associated with lower incidences of endometrial cancer and contralateral breast cancer occurrence (5). Although these new aromatase inhibitors are shown to be very potent and specific, the structural basis of drug recognition by aromatase has remained elusive because the three-dimensional structure of this enzyme is not yet determined.


Figure 1
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  		Fig. 1. Structures of Aromatase Substrate Androstenedione, Product Estrone, Steroidal Inhibitor Exemestane, and Nonsteroidal Inhibitors Letrozole and Anastrozole

 
The detailed structural characterization of aromatase purified from human placenta has been hampered by its membrane-bound character and heme-binding instability. In prokaryotes, P450s are soluble proteins. The first three-dimensional structure of cytochrome P450 species that has been solved is the soluble P450cam from Pseudomonas putida by Poulos et al. (6). Various theoretical structural models of aromatase have been generated based on these soluble bacterial cytochrome P450s (7, 8, 9, 10, 11, 12, 13). However, the percentage sequence identity between aromatase and these bacterial P450 templates range from 13–18% (14). The first crystal structure of the mammalian P450 enzyme, P450 2C5 from rabbit, was solved by Williams et al. (15). The N-terminal transmembrane domain was deleted and some internal mutations were introduced in P450 2C5 to facilitate protein solubility and crystallization. Using a similar approach, they determined the first crystal structure of human P450 enzyme, P450 2C9 (16). These newly solved crystal structures of mammalian cytochrome P450s offer more reliable templates than bacterial templates for modeling aromatase. A structural model of aromatase was generated by using rabbit CYP2C5 as a template in our laboratory, and this model has been helpful in better understanding the structure-function relationship of aromatase (14). Recently, a theoretical three-dimensional model of aromatase was built based on the crystal structure of human CYP2C9 (17). Although the overall sequence identity in the final alignment was only 20%, the percentage of identical amino acids in the core of the protein (residues in a sphere of 4 radius Å from the heme) was 54%.

The detailed structure-function studies of aromatase need adequate amounts of purified active enzyme. A number of attempts have been devoted to the expression and purification of human recombinant aromatase (18, 19, 20, 21, 22). Unfortunately, these earlier studies were not successful in obtaining large amounts of the purified active enzyme due to its hydrophobic nature and instability. Kagawa et al. (20) reported the production of a recombinant aromatase (NmA264R) with improved yield in Escherichia coli based on coexpression with molecular chaperones GroES/GroEL. By modifying our previous approach (19), we have recently achieved the expression of a structurally stable and functionally active human aromatase in E. coli and have successfully purified the enzyme. Using this purified preparation, the detailed structure-function relationships of aromatase have been studied by UV/Vis spectral analysis and proteomic analysis. In addition to experiments using recombinant aromatase preparation, we have performed site-directed mutagenesis in a mammalian expression system and carried out computer modeling based on results generated from our and other laboratories.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
Conclusion
 MATERIALS AND METHODS
 REFERENCES
 
Purification of Recombinant Human Aromatase NmChAro
The original full-length CYP19 cDNA was isolated by Pompon et al. (18). To improve its solubility, the amino-terminal transmembrane domain (residues 1–39) was replaced by a short positively charged, hydrophilic polypeptide MAKKTSSKGR, identical with that employed for the expression and crystallization of CYP2C5 (15, 23), CYP2C8 (24), and CYP2C9 (16). A six-histidine tag was introduced to the C terminus to facilitate purification. The modified cDNA (N-MAKKTSSKGR, C-6xHis Del-39) was cloned into the pET3b vector. The resulting enzyme is designated NmChAro.

NmChAro was solubilized from E. coli membrane with detergent (Tween 20)-containing buffer in the presence of its substrate, androstenedione, and purified using Ni Sepharose, hydroxyapatite, and Superdex 200 (Amersham, Piscataway, NJ) FPLC chromatography columns. Superdex-200 chromatography was used for the last purification step to remove detergent and any aggregates. Figure 2Go, A and B, summarizes the results of each step of the purification procedure. The purified NmChAro appeared as a single band with molecular mass of 55 kDa (55,446 based on its amino acid sequence). The aromatase activity was recovered with a 77% overall yield, having a specific activity of 68.2 nmol/mg·min.


Figure 2
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  		Fig. 2. Purification and Characterization of NmChAro

A, SDS-PAGE of purification of NmChAro, detected by coomassie blue. The lanes shown are the molecular weight markers (Std), crude extract by 0.1% Tween 20 (Ext), the nickel column pass-through (Pt1), the nickel column 20 mM imidozole washing fraction (W1), the nickel column 50 mM imidozole washing fraction (W2), the nickel column elute (Elu-N), the hydroxylapatite column pass-through (Pt2), the hydroxylapatite column elute (Elu-H), the Superdex-200 column elute (Elu-S) and the molecular weight markers (Std). B, Purification of NmChAro from 1 liter E. coli culture. C, Enzyme kinetic analysis of purified NmChAro. This analysis was performed using the tritiated water release method with [1ß-3H]androstenedione concentrations from 10 nM to 500 nM. Each point represents the mean of triplicate experiments.

 
Characterization of Purified NmChAro
Analysis of aromatase activity over an extended period showed that the purified NmChAro was very stable, retaining greater than 90% of initial activity at 4 C after 3 wk in 25 mM Na-HEPES buffer (pH 7.4) containing 0.15 M NaCl, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol (DTT), and 10 µM androstenedione. However, after a freeze-thaw cycle, only approximately 75% of initial activity maintained.

The Km (Michaelis-Menten constant) and Vmax (maximum velocity) values of NmChAro were estimated to be 301 nM and 130 nmol/mg·min for androstenedione (Fig. 2CGo). Kinetic parameters obtained in this study are compared with values previously reported in the literature (Supplement A, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). The Km value of 301 nM is higher than previous reports of references21 , 25 , 26 , and 27 ; however, it is comparable with that of recombinant aromatase in Kagawa et al. (20). Perhaps the variations are due to differences in the assay conditions used. Our recombinant aromatase has a maximal turnover number of 8.3 min–1, comparable with those previously reported in references20 , 21 , 26 , and 27 (Supplement A).

Spectral Characteristics of NmChAro
In the oxidized state, the Soret maximum of the purified NmChAro-androstenedione complex was at 394 nm (Fig. 3AGo), indicating a typical substrate binding spectrum (see discussion below). The absorption maximum of the ultraviolet region was at 278 nm, and the A278/A394 ratio (protein vs. heme) was 1.03 (Fig. 3AGo). Figure 3BGo shows the time course-reduced carbon monoxide (CO)-difference spectra of the purified NmChAro-androstenedione complex. The absorbance difference between 450 nM and 490 nM was 0.043, indicating the P450 concentration is 0.47 µM (Fig. 3CGo). The P450 content is 15.7 nmol/mg of protein, which is 87% of the expected value (18 nmol P450/mg protein) as calculated from the molecular weight of pure aromatase protein.


Figure 3
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  		Fig. 3. Spectral Properties of the Purified NmChAro

A, Oxidized spectrum in the ultraviolet-visible region. The sample contained 0.54 µM (30 µg/ml) NmChAro in the presence of 4 µM androstenedione. B, Time-course reduced CO-difference spectra. The sample was scanned every minute after addition of sodium dithionite into the CO-saturated sample. Because the reduction of NmChAro by sodium dithionite was slow, the absorbance at 450 nm reached a maximum in about 10 min. C, Reduced CO-difference spectrum. D, Oxidized spectra in the ultraviolet-visible region of purified NmChAro induced by androstenedione and inhibitors. E, Spectral analysis in the UV/Vis region of purified NmChAro at zero time after incubation with androstenedione and inhibitors at 37 C for 10 min in the presence of human NADPH-P450 reductase plus NADPH. F, Spectra in the ultraviolet-visible region of reacted NmChAro after 30 min incubation on ice. In panels D–F, 1) ligand-free NmChAro; 2) NmChAro-androstenedione (0.36 µM:1 µM) complex; 3) NmChAro-exemestane (0.36 µM:1 µM) complex; and 4) NmChAro-letrozole (0.36 µM:1 µM) complex.

 
Using the purified preparation, the UV/Vis absorption spectra induced by the binding of substrate and inhibitors were investigated (Fig. 3DGo). In the oxidized state, ligand-free NmChAro exhibited a Soret absorption maximum at approximately 420 nm. This is associated with the low-spin state of the Fe3+ with a water molecule as a sixth proximal axial ligand. When bound to androstenedione, the complex produced a type I binding spectrum, characterized by a reduction in the Soret band at 420 nm and a corresponding absorption maximum at 394 nm. This is indicative of a displacement of iron-ligated water molecule by androstenedione, changing the Fe3+ from a low-spin to a high-spin state. The type I binding spectrum was also observed in the NmChAro-exemestane complex, as expected for a pseudo-substrate. The type II binding spectrum was observed in the NmChAro-letrozole complex in this experiment, identified by an increase in the absorption at 422 nm and a decrease at 394 nm. This spectral change is associated with the interaction of letrozole that binds directly to the Fe3+, displacing the water molecule as the sixth axial ligand, and increasing in the low-spin character of the Fe3+.

The type I binding spectrum induced by exemestane suggests that exemestane binds to the substrate-binding site during the first step of inhibition. It is well known that mechanism-based inhibitors cause time-dependent inactivation of aromatase only in the presence of cofactors such as NADPH (28). To better define exemestane as a mechanism-based inhibitor, exemestane was incubated with purified NmChAro in the presence of human NADPH-P450 reductase and NADPH. The reaction mixture was kept at 4 C subsequent to the 10 min incubation at 37 C, and the time course spectra were recorded. Gonzalez and Piferrer (29) previously showed that aromatase activity at 4 C is around 20% of the maximum at 37 C. We assume that the reaction slow down significantly at 4 C. The spectrum of NmChAro reduced by NADPH exhibited a greatly increased absorption between 300 and 400 nm, which prevented the examination of the absorption at 394 nm. However, the absorption at 420 nm could be recorded. At zero time, there was no peak at 420 nm in the reaction mixture with exemestane or androstenedione, whereas in the reaction mixture of ligand-free enzyme or with letrozole, an absorption peak at 420 nm was observed (Fig. 3EGo). The absorption at 420 nm appeared in the reaction mixture with androstenedione after a 30 min incubation on ice (Fig. 3FGo). However, the reaction mixture with exemestane failed to recover the 420-nm peak (Fig. 3FGo) even after overnight incubation on ice. These results indicate that after the aromatization of androstenedione, estrone releases from the enzyme, allowing a water molecule to re-ligate to iron, switching it back to a 6-fold coordination state. In contrast, acting as a mechanism-based inhibitor, exemestane (or its intermediates) fails to release once it binds to the enzyme.

Proteolytic Analysis of NmChAro
Based on the published crystal structures of P450s, the overall fold is maintained in all P450s (30). There are two domains in P450s: the {alpha}-domain is associated with the catalytic center, and the ß-domain is associated with substrate recognition and the access channel (31). The heme group is sandwiched between the helices I and L, whereas the helices B, C, F, and G contribute to substrate recognition (17).

To study the three-dimensional folding of human aromatase, we have performed time-dependent proteolytic digestion of purified NmChAro. Purified NmChAro preparation was incubated with trypsin in nondenaturing conditions for various time periods, and the proteolytic products were analyzed by SDS-PAGE (Fig. 4AGo). As a control, digestion of denatured protein (preheat NmChAro preparation in a 37 C water bath for 2 h, a P450 peak shifted to a P420 peak in reduced CO-difference spectra) produced different fragmentation patterns by SDS-PAGE analysis (data not shown). According to the crystal structures of P450s complexed with their ligands, conformational changes within the active site can be induced by the binding of substrate or inhibitor (30). To demonstrate whether binding of ligands to aromatase induces conformational changes, the proteolytic products of NmChAro complexed with androstenedione, exemestane, or letrozole were compared with those of the ligand-free enzyme. Androstenedione, exemestane, and letrozole protect aromatase from proteolysis by trypsin (Fig. 4BGo). These results demonstrate aromatase adopts a more compact conformation when bound to ligands.


Figure 4
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  		Fig. 4. Proteolytic Digestion of Purified NmChAro by Trypsin

A, SDS-PAGE of time-course trypsin-digested NmChAro detected by coomassie blue. Purified NmChAro was incubated with trypsin (NmChAro:trypsin = 100:1) in the absence of ligands in a 37 C water bath for 5, 10, 20, and 30 min. The lanes shown are the molecular weight markers (Std), 10 µg undigested purified NmChAro (Un-D), 0.1 µg trypsin (Tryp), and 5, 10, 20, and 30 min trypsin-digested NmChAro. B, SDS-PAGE of trypsin-digested NmChAro detected by coomassie blue. Unliganged or liganded NmChAro were incubated with trypsin (NmChAro:trypsin = 20:1) respectively in a 37 C water bath for 30 min. The lanes shown are the molecular weight markers (Std), 10 µg undigested purified NmChAro (Un-D), 1µg trypsin (Tryp), trypsin-digested unliganded NmChAro (Un-L), trypsin-digested NmChAro-androstenedione (1:1) complex (And), trypsin-digested NmChAro-exemestane (1:1) complex (Exe), and trypsin-digested NmChAro-Letrozole (1:1) complex (Let).

 
The hydrolyzed peptide mixtures were then analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to determine the fragmentation patterns (Supplement B). The experimentally measured peptides’ mass-to-charge values were compared with those of predicted potential peptides based on cleavage sites calculated by PeptideCutter (ExPASy, Swiss Institute of Bioinformatics, Switzerland). We were able to identify peptides with masses ranging from 870-6094 Da.

The fragmentation patterns were aligned with the amino acid sequence of aromatase as shown in Fig. 5Go. In the time-course digestion study, it was found that trypsin cleavage started from the carboxyl terminus of NmChAro (i.e., the {alpha}-domain). After a 5-min digestion, trypsin cleaved the peptide bonds in the flanking loops of the L helix, while leaving the L helix intact. These regions are on the surface of the three-dimensional model from Favia et al. (17). After a 10-min digestion, further cleavage occurred in the C terminus, including the L helix itself. After a 20-min digestion, more peptides were detected within the C terminus. Moreover, cleavage occurred among the B-C loop, C and D helices, and the C-D turn. After a 30-min digestion, besides previous fragmentation products, cleavage also occurred in the ß-domain of the protein, including the B helix, ß-sheets 1-4, 1-3, 2-1, and 2-2, and the N terminus (the transmembrane segment, removed for solubility, is not included).


Figure 5
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  		Fig. 5. Fragmentation Patterns of NmChAro by Trypsin Are Aligned with the Amino Acid Sequence of Aromatase

In this recombinant aromatase (NmChAro), the amino-terminal transmembrane domain (residues 1–39) was replaced by a polypeptide MAKKTSSKGR, and a six-histidine tag was introduced to the C terminus. The predicted secondary structure of the model proposed by Favia et al. (17 ) is indicated by green helix ({alpha}-helix) and pink arrow (ß-sheet) above its primary sequence. Peptides detected by MALDI-TOF-MS are underlined (yellow: 5 min digestion; red, 10 min digestion; purple: 20 min digestion; blue, 30 min digestion). Peptides that are protected from fragmentation in the presence of androstenedione or exemestane are highlighted within a blue colored box.

 
The fragmentation patterns were aligned onto the three-dimensional model from Favia et al. (17) as shown in Fig. 6Go and supplement C. This time-dependent digestion study shows that cleavage starts from the surface of the {alpha}-domain of the enzyme (except the F helix and F-G loop), followed by digestion of the center of the {alpha}-domain, and lastly in the compact ß-domain. The presence of the intact F helix and F-G loop after a 30-min digestion is thought to result from the direct intermolecular interactions among them in the symmetric dimer. Some P450s have demonstrated dimer formation in crystals through the interaction of the helices F and G region, such as P450 2C5 (15) and 2C8 (24). In addition, our gel-filtration experimental results demonstrated that a dimer of NmChAro forms in solution (results not shown).


Figure 6
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  		Fig. 6. A Stereo View of the Time-Dependent Fragmentation Patterns of Aromatase Protein that Was Digested by Trypsin (Yellow, 5-Minute Digestion; Red, 10-Minute Digestion; Green, 20-Minute Digestion; Blue, 30-Minute Digestion)

The cleavage sites of lysine or arginine are shown in sticks. The heme (orange) is in the catalytic center of the enzyme, and the iron (shown in spheres) is located in the center of the heme.

 
Interestingly, the ligand-protection digestion showed that the B and C helices and the B-C loop (that contains I133 and F134, whose importance will be discussed below) resist fragmentation in the androstenedione-NmChAro and exemestane-NmChAro complexes as highlighted in the blue box in Fig. 5Go, whereas this portion hydrolyzes easily in ligand-free NmChAro and Letrozole-NmChAro complex. This might be due to the dramatic reshaping in this region when the enzyme binds to substrate or steroidal inactivator. Overall, our proteomic results reveal a three-dimensional overall folding of human aromatase, which is compatible with the three-dimensional model from Favia et al. (17).

Reaction Intermediate Studies
The biosynthesis of estrogen from androgen by aromatase proceeds in three hydroxylation steps. The first and second hydroxylations occur at the 19-methyl group by the classical enzyme-mediated hydrogen atom abstraction-hydroxyl radical rebound mechanism (32, 33). Various mechanisms (34, 35, 36, 37) have been proposed to explain the mysterious third hydroxylation step, which cleaves the C10-C19 bond, resulting in aromatization of the steroid A-ring and release of formic acid. Model reaction studies, including data from our group, support mechanism that an enolization of a carbonyl group at C-3 toward the C-2 position would be a prerequisite for the cleavage of the C10-C19 bond (38, 39). Recently, Hackett et al. (40) further demonstrated that 1ß-hydrogen atom abstraction by an iron-oxo intermediate from substrates in the presence of the 2, 3-enol encounters strikingly low barriers (5.3–7.8 kcal/mol), whereas barriers for this same process rise to 17.0–27.1 kcal/mol in the keto tautomer. The residues involved in the enolization of C-3 toward C-2 would be critical for the final catalytic step, although these critical residues have not yet been determined.

Our previous data showed that more 19-hydroxyandrostenedione (19-ol) intermediate was generated by both S478A and S478T than the wild-type (WT) enzyme, and significantly more 19-ol and 19-oxoandrostenedione (19-al) intermediates were detected for both mutants H480K and H480Q than for the WT enzyme (39). Our results indicate that these mutations affect the final aromatization step that lead to the accumulation of 19-ol and 19-al intermediates.

To better understand the molecular basis for the aromatization reaction, we further determined the reaction intermediate profiles of 10 mutants (K119R, K119N, E129D, E129Q, F134Y, F134L, I305A, I305V, H475A, and H475A) and compared those to that of WT aromatase. These five amino acids were thought to be located at the substrate binding pocket, according to our computer model based on mammalian P450 2C5 as the template (14). These mutants were prepared and analyzed using a mammalian cell expression system. The expression levels of WT aromatase and the ten mutants were similar in transfected Chinese hamster ovary (CHO) cells by immunoprecipitation analysis (Supplement D). The catalytic properties of the aromatase mutants have been determined using an in-cell aromatase assay, and the apparent Km and Vmax values are shown in supplement E. The reaction intermediate profiles showed no significant differences of 19-ol or 19-al intermediates between the WT and each mutant (data not shown). Interestingly, significant amounts of 19-nor-4-androstene-3, 17-dione (19-nor) were produced by F134Y (Fig. 7AGo), approximately 4-fold higher than that of WT levels, and 19-nor was suggested to be artifactually derived from 19-oic-androsteneione as a result of degradation (41).


Figure 7
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  		Fig. 7. Site-Directed Mutagenesis: Reaction Intermediate Analysis and Inhibitory Profile Analysis of Aromatase and Its Mutants

A, Reaction intermediate analysis of WT aromatase and the F134Y mutant. Results are shown as means ± SD (n = 3). B, Inhibition analysis of human aromatase by exemestane in CHO cells: dose response of exemestane on WT, and mutants E302D, D309A, and S478T. Each analysis was performed in triplicate, and bars indicate SD. C, Inhibition analysis of human aromatase by exemestane in CHO cells: time course of WT, and mutants E302D, D309A, and S478T to 20 nM exemestane. Each analysis was performed in triplicate, and bars indicate SD.

 
F134 has been proposed to play an important role in enzyme catalysis (10). Our new mutagenesis data showed the catalytic activities of F134L were significantly lower than F134Y (Supplement E), indicating an aromatic amino acid is needed at this position. The accumulation of 19-nor in F134Y also indicated a hydrophobic residue is necessary. We have undertaken an extensive investigation of the interaction of different aromatase inhibitors with these two mutants, for the determination of the binding characteristics of different steroidal and nonsteroidal inhibitors. Mutants F134L and F134Y greatly modified the binding affinities of all inhibitors tested (data not shown). These results indicate that F134 is critical for the substrate binding and enzyme catalysis.

Molecular Basis for Exemestane Irreversible Inhibition
Exemestane is an androstenedione derivative and has been shown to be a mechanism-based aromatase inactivator (28, 42). However, the molecular nature for the interaction of this compound with the enzyme is still unclear.

Compounds lacking a C19 methyl group did not cause a time-dependent decrease in aromatase activity (43). Incubation of another suicide inhibitor androst-4-ene-3, 6, 17-trione (AT) with human placental microsome yielded the 19-hydroxy-AT and 19-oxo-AT (44). These results lead us to propose that a reactive electrophile produced from the hydroxylation at the C19 of exemestane binds to the active site in an irreversible manner, resulting in inactivation of aromatase.

To better understand the molecular basis for the interaction of exemestane with aromatase, we first performed inhibitory profile analyses of the WT and 12 mutants (K119R, I229V, I133Y, I133W, F134Y, E302D, I305A, A306G, D309A, T310S, S478T, and H480Q) transfected in CHO cells. Our results showed that exemestane was significantly less potent in inhibiting the mutants, E302D (IC50 = 96 nM), D309A (IC50 = 40 nM) and S478T (IC50 = 64 nM), than WT aromatase (IC50 = 22 nM) (Fig. 7BGo). The IC50 value of 22 nM for WT aromatase is similar to the inhibition constant value of 26 nM reported by Giudici et al. (42).

Next, a time course study of the inhibition of WT aromatase, and mutants E302D, D309A, and S478T by exemestane was performed. Aromatase activity was measured after various incubation periods with exemestane. A definitive time-dependent inhibition of WT aromatase by exemestane was observed, as expected for a suicide inhibitor (Fig. 7CGo). The time required for 50% inhibition was also similar to the t1/2 value of 13.9 min reported by Giudici et al. (42). However, time-dependent inhibition of these three mutants could not be clearly demonstrated (Fig. 7CGo). These results indicate that E302, D309, and S478 may participate in the mechanism responsible for suicide inhibition of aromatase by exemestane. These results will be discussed below.

Substrate Binding and Catalytic Mechanism
In an effort to better understand the molecular mechanism of the aromatization reaction and to investigate the roles of active site amino acids discussed above, androstenedione was docked into the three-dimensional model from Favia et al. (17). In this model, the heme iron is ligated by a conserved cysteine (Cys437) and the propionates of the heme interact with the side chains of R115, W141, R145, R375, and R435. The active site pockets from this model and previous models (9, 10) are in agreement except in the region of the B'-C loop and the ß-4 sheet. In this model, I125-P138 of the B'-C loop penetrates into the interior of the molecule so that I132-F134, rather than H128-K130 in the model from Graham-Lorence et al. (9), are located in or adjacent to the active site. E129 and K130 are now thought to stabilize this turn structure: E129 forms a hydrogen bond with N135 and K130 forms a hydrogen bond with N136. In addition, sheet ß4 protrudes into the active site, placing L477-H480 above the active site pocket, whereas L473-D476 is further away from the active site. Instead of directly interacting with the substrate in the active site pocket, L473-D476 is now thought to localize within the putative substrate access channel.

The substrate binding site of aromatase was selected according to the x-ray crystal structure of cytochrome P450eryf with androstenedione bound (PDB ID: 1EUP), and further refined by Tripos LITHIUM (currently marketed as Benchware 3D Explorer; Tripos, St. Louis, MO). As shown in Fig. 8Go, the C19 methyl group, which undergoes the first and second hydroxylation reactions, is centered over the heme-bound iron atom, and is 3.3 Å from T310. It is believed that the conserved threonine (T310) plays an important role in the oxygen-oxygen bond cleavage. D309, S478, and H480, which flank the A-ring of androstenedione (Fig. 8Go), form a catalytic triad consistent with our previously proposed mechanism (39). D309 is thought to play important roles in all three hydroxylation steps. The first and second hydroxylations at the C19 methyl group are consistent with any other P450-catalyzed hydroxylation (Fig. 9AGo). Molecular oxygen is bound by the reduced heme iron, forming peroxide. After forming the iron-oxo intermediate facilitated by T310, T310 is immediately reprotonated by the neighboring D309. These two hydroxylation steps produce 19-ol and 19-diol, respectively. It is unclear whether 19-diol or 19-al is the true intermediate for the second hydroxylation step. Studies from Hackett et al. (40) do not support the dehydration of the 19-diol before the final catalytic step. Here we assume that the 19-diol remains hydrated until the final decarbonylation. The detection of 19-al from our reaction intermediate analysis may be due to dehydration from the unstable form of 19-diol under experimental conditions.


Figure 8
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  		Fig. 8. Clamping Mechanism of the Substrate Androstenedione Binding Provided by the Heme, I Helix, B'-C Loop, and the ß-4 Sheet

The C19 group, which undergoes the hydroxylation reactions, is centered over the iron. I133 and F134, which are located in the B'-C loop, interact with substrate through Val der Waals forces, and the structure of the B'-C loop is stabilized by the H-bonds between E302 and the backbone nitrogen of these two residues. D309, T310, S478, and H480, flank the A-ring of substrate, they are involved in the aromatization reaction. The substrate is also stabilized through the H-bonds between S478 and C3-keto group.

 

Figure 9
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  		Fig. 9. Molecular Mechanism of the Aromatization Reaction

A, Proposed mechanism of the first and second hydroxylation steps. T310 is involved in oxygen-oxygen bond cleavage; D309 provides proton for T310. These two hydroxylation steps produce 19-ol and 19-diol, respectively. B, Proposed mechanism of the aromatization step. D309 and T310 play the same roles in the iron-oxo intermediate formation as in the first and second hydroxylation steps. During enolization, D309 extracts 2ß-hydrogen, helped by H480; S478 donates a proton to 3-keto. 1ß-Hydrogen is abstracted from the 2, 3 enol by iron-oxo intermediate in the presence of gem-diol, followed by deformylation.

 
D309 is also believed to extract 2ß-hydrogen during the subsequent aromatization reaction (14), which is helped by H480, forming a salt bridge with D309. In addition, S478 is located near the A-ring, forms a hydrogen bond with the C-3 keto oxygen to stabilize the substrate (Fig. 8Go), and may donate a proton to the C-3 keto oxygen during enolization (Fig. 9BGo). The mechanism of the aromatization step proposed in this study is similar to that in Graham-Lorence et al. (9), except for the amino acid residues for proton donation to the 3-keto group of the substrate. S478 is thought to be the residue in this study, whereas K473 or H475 in the analysis by Graham-Lorence et al. (9). Based on our mutagenesis results, reaction intermediate studies, and computer modeling, S478 is important in both positioning androgen and participating in aromatization reaction. Because D309 is thought to be involved in all three hydroxylation steps, it is proposed that either the side chain of D309 is flexible or the substrate moves, repositioning the side chain of D309 to face the C-2 carbon during enolization.

The B'-C loop also appears to play an important role in orienting the substrate. The D-ring is anchored by Val der Waals forces from I133 and F134, particularly the aromatic side chain of F134 on the C-18 methyl group. I133 is important in providing hydrophobic interactions with the substrate, stabilizing it in the active site pocket. We believe that the aromatic {pi}-cloud of F134 interacts with C18, rather than C19 as believed in the model proposed by Auvray et al. (10), and this positions the substrate inside the active site. When F134 is mutated to a tyrosine residue, the polar side chain of tyrosine might decrease its interaction with the substrate, affecting the position of C-2 with D309 and retarding the enolization of C-3 toward C-2 in the aromatization step. Therefore, the accumulation of 19-nor in F134Y would be a result of degradation from 19-oic-androsteneione, due to a hindrance in the 2,3-enolization.

Implications for the Mechanism of Exemestane Irreversible Inhibition
To investigate the molecular mechanism of irreversible inhibition, exemestane is docked into the same site as androstenedione. As shown above, E302, D309, and S478 may participate in the mechanism of suicide inhibition of aromatase by exemestane. We will focus on the molecular interaction of these three residues with exemestane.

The function of E302 is ambiguous. Mutant E302L was found to be unstable and inactive in our group (7), as well as mutant E302A (10). Graham-Lorence et al. (45) also reported that mutant E302D had 30% of the activity of the WT enzyme, and mutant E302Q had minimal activity. These mutagenesis results suggest that the acidic group at this position is important, and lead us to propose that E302 interacts with the substrate and is involved in the first and second hydroxylations (39). However, E302 is now judged as being too far from the substrate. Our present results from the irreversible inhibitory analysis of exemestane emphasize the role of E302, which participates in the mechanism of suicide inhibition of aromatase by exemestane. Furthermore, E302 was aligned with D293 of the P450 2C9 template. In the crystal structure of P450 2C9, D293 forms a hydrogen bond with the backbone nitrogen of I112. In the present model, we proposed E302 forms a hydrogen bond with the backbone nitrogen of I133 and F134 of the B'-C loop, preventing this important loop from drifting away. It is consistent with other groups (9, 17) that E302 maintains the structure of the active site cavity, although they have suggested that E302 forms a salt-bridge with K130. The mutant E302D would be too far to form a hydrogen bond with the backbone nitrogen of I133 and F134, and therefore the critical penetrating loop structure of the B'-C loop might not be stabilized. This minimizes the interaction between I133 and F134 with exemestane (or its intermediates), so that exemestane (or its intermediates) may not be stabilized in the active site pocket.

As discussed above, the function of D309 is in agreement among different groups. The mutant D309A was remarkably resistant to a great number of inhibitors (13, 46). Interestingly, the present inhibitory analysis of exemestane shows that D309A fails to maintain a time-dependent inhibition of exemestane. Furthermore, previous data from our lab showed that neither 19-hydroxyandrostenedione nor 19-oxoandrostenedione intermediates were produced in D309A and D309N mutants (7). We propose that the D309A mutant retards the hydroxylation of C19 of exemestane, so that it fails to produce exemestane intermediates to cause a time-dependent inhibition. Based on our analysis, we also hypothesize that D309 is involved in the irreversible binding of exemestane intermediates.

In the exemestane-bound model, S478 is located near the A-ring, forming a hydrogen bond with the C-3 keto oxygen to stabilize exemestane (or its intermediates). The larger side chain of the mutant T478 may cause conformational conflicts with exemestane; therefore, the threonine side chain must rotate, and as a result the hydrogen bond is lost. This weaker interaction between exemestane (or its intermediates) and the mutant enzyme causes exemestane (or its intermediates) to be less stable in the active site pocket, and possibly accounts for the loss of irreversible inhibition.

Therefore, we propose the mechanism of irreversible inhibition of exemestane. Firstly, exemestane binds to the substrate-binding site, particularly by Val der Waals forces from I133 and F134 of the B'-C loop and the hydrogen bond formed between S478 and C-3 keto group. Exemestane is then converted to reactive intermediates by heme through the hydroxylation of C19 group, helped by D309. Finally, the intermediates irreversibly bind to the enzyme cause suicide inhibition, in which D309 may be involved.


   Conclusion
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 Conclusion
MATERIALS AND METHODS
 REFERENCES
 
It is important to solve the three-dimensional structure of human aromatase, which would further enable the application of structure-based design of potent and selective inhibitors. However, to date, aromatase has resisted structural elucidation by x-ray crystallography due to its membrane-bound character and heme-binding instability. In this report, structurally stable, functionally active, and successfully purified recombinant human aromatase provides a promising perspective for crystallography, although further internal modifications on the F-G loop may be necessary.

While we are expecting to solve the x-ray structure of aromatase, detailed structure-function analysis using experimental methods described in this report has provided useful structural information. We propose a new clamping mechanism of substrate/exemestane binding to the active site that involves the heme, I helix (between E302 and T310), B'-C loop (containing I133 and F134), and the ß-4 sheet (containing S478 and H480), particularly the clamping motion of the B'-C loop when the enzyme binds to androstenedione or exemestane. This new clamping mechanism explains most of the site-directed mutagenesis results from this and other laboratories, and helps us to understand the molecular action of substrate/drug recognition by aromatase. These studies have produced valuable information to design the next generation of aromatase inhibitors for the prevention and the treatment of estrogen-dependent breast cancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 Conclusion
 MATERIALS AND METHODS
REFERENCES
 
Expression and Purification of NmChAro
The E. coli BL21 (DE3) strain was used for the expression of NmChAro. Bacteria was harvested, incubated on ice for 30 min with 0.5 mg/ml lysozyme in buffer A [100 mM potassium-phosphate buffer (pH 7.4), 20% glycerol, 1 mM DTT, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 µM androstenedione], and disrupted by sonication on ice (Branson Sonifier 450, ~70% full power, 3 x 1 min). NmChAro was isolated from the pelleted membranes with buffer B (buffer A containing 0.1% Tween 20 and 0.5 M NaCl), and purified by metal-ion affinity chromatography (Ni Sepharose 6 Fast Flow; Amersham). After elution of NmChAro with a linear imidozole gradient from 50 mM to 300 mM in buffer B, the red fractions were pooled, desalted, applied to a hydroxyapatite (Bio-Rad) column for the elimination of minor contaminants, and eluted with a linear gradient of 0–1 M NaCl in buffer C (buffer A containing 0.1% Tween 20). Purified NmChAro was loaded on a gel-filtration column (Superdex 200) to remove aggregates and Tween 20 detergent, eluted with buffer D [25 mM Na-HEPES buffer (pH 7.4), 0.15 M NaCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, 10 µM androstenedione], and concentrated using a centrifugal device (Ultra-15 30K; Millipore, Billerica, MA). Purified and concentrated NmChAro was stored in buffer D. For the ligand-free NmChAro preparation, the sample was eluted from a gel-filtration column in buffer D without adding its substrate androstenedione.

Aromatase Activity Assay
Aromatase activity was determined according to the published tritiated water-release method (19), with a modification. The standard in vitro assay was reconstituted with 100 nM human NADPH-P450 reductase (BD Biosciences, Franklin Lakes, NJ) in a 500-µl reaction buffer containing 67 mM potassium phosphate (pH 7.4), 0.1% BSA, 10 µM progesterone, and 500 nM [1ß-3H]androstenedione at 37 C in a shaking water bath for 20 min. The incubation was initiated by the addition of 300 µM of NADPH, and terminated by the addition of 500 µl 20% trichloroacetic acid. The reaction was mixed with charcoal-dextran to remove any trace amount of unreacted substrate. After centrifugation of the mixture, the radioactivity of the supernatant was counted by a liquid scintillation counter (LS 6500; Beckman Coulter, Inc., Fullerton, CA). Protein concentrations were determined by the Bradford assay method (47).

In the in-cell aromatase assay, aromatase-transfected CHO cells were seeded in six-well plates, and 1 ml serum-free media containing 100 nM [1ß-3H] androstenedione, as well as 1 µM progesterone, were added to each well. To determine protein concentration, cells remaining in each well were solubilized with 0.5 M NaOH and subjected to the Bradford assay method.

UV/Vis Spectral Analysis
The absorption spectra of the purified preparation were measured by a UV-1700 PharmaSpec UV-Vis Spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD) with a quartz cuvette having a 1-cm optical path. To measure the reduced CO-difference spectra, the samples were aliquoted into two cuvettes, followed by a baseline spectrum recording between 650 and 350 nm. After bubbling carbon monoxide slowly into the sample cuvette, a few milligrams of sodium dithionite was added to the sample cuvette, mixed, and the spectrum difference was recoded. The specific P450 content was determined by Beer’s Law, with a molar extinction difference of 91 mM–1·cm–1 between 450 and 490 nm.

Proteomic Analysis
For the time-course digestion, purified aromatase preparations were incubated with trypsin (NmChAro:trypsin = 100 µg:1 µg) in the absence of ligand in a 37 C water bath for 5, 10, 20, and 30 min. For the ligand-protected digestions, preparations were incubated with different ligands (NmChAro:ligand = 1:1) for 30 min on ice, then the unliganded or liganded samples were treated with trypsin (NmChAro:trypsin = 20 µg:1 µg) in a 37 C water bath for 30 min. The digestions were terminated by the addition of acetic acid until the pH dropped below 4, then analyzed by the MALDI-TOF MS.

MALDI-TOF MS
Samples were desalted and concentrated with ZipTip C18 pipette tips (Millipore), eluted with 50% acetonitrile solution, mixed with MALDI matrix {alpha}-cyano-4-hydroxy-cinnamic acid ({alpha}-cyano), which is suitable for small peptides with peptides’ mass-to-charge below 10,000, spotted on a MALDI plate, and directly applied on MALDI-TOF MS (Voyager-DE STR; PerSeptive Biosystems Inc., Framingham, MA).

Site-Directed Mutagenesis
The mutant cDNAs were generated by the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using the WT aromatase expression plasmid pHß-Aro (48) as template, then transformed into DH5{alpha} cells. All mutations were verified by DNA sequencing. For stable transfections, mutant plasmids were transfected into CHO cells using lipofectin (Life Technologies, Inc., Rockville, MD). After 2 wk of G418 selection, transfected cells were maintained in media containing 600 µg/ml G418.

Reaction Intermediate Analysis
The aromatase or mutant transfected cells were incubated with 100 nM [1ß-3H]androstenedione. After a 1-h incubation, the reaction media were extracted with an equal volume of chloroform, and the chloroform solvent was removed by vacuum evaporation. The sample was dissolved in 100 µl of acetonitrile; a 50-µl aliquot was mixed with 10 µl of internal standards (200 nM each of 19-ol A, 19-al A, 19-nor A, and androstenedione). The reaction intermediates were separated by reverse-phase HPLC on a C18 column (218TP54; VYDAC, Hesperia, CA). One-milliliter fractions were collected manually, and 1-ml aliquots were counted in 3 ml of Scintisafe liquid scintillation fluid (Fisher Scientific, Pittsburgh, PA). Retention times of 19-ol A, 19-al A, 19-nor A, and androstenedione were determined based on the internal standards at 9, 16, 25, and 32 min, respectively. The radioactivity associated with each peak was used to calculate the amount of each intermediate.


   ACKNOWLEDGMENTS
 
We are grateful to Prof. Mary K. Young (City of Hope) for helping with MALDI-TOF-MS, as well as to Prof. Thomas Poulos, Dr. Yergalem Meharenna (University of California, Irvine), Prof. Terry Lee, Mr. Roger Moore, Dr. Leila Su, Ms. Kim Karlsberg, and Ms. Selma Masri (City of Hope) for valuable discussion.


   FOOTNOTES
 
This work was supported by California Breast Cancer Research Program [11GB-0125 (to Y.H.)] and National Institutes of Health [CA44735 (to S.C.), ES08528 (to S.C.), and CA33572 (the City of Hope Cancer Center grant)].

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 9, 2006

Abbreviations: 19-al, 19-Oxoandrostenedione; AT, Androst-4-ene-3, 6, 17-trione; CO, carbon monoxide; CHO, Chinese hamster ovary; DTT, dithriothreitol; MALDI-TOF MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; NADPH, reduced nicotinamide adenine dinucleotide phosphate; 19-nor, 19-nor-4-androstene-3, 17-dione; 19-ol, 19-hydroxyandrostenedione; WT, wild type.

Received for publication July 7, 2006. Accepted for publication October 30, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 Conclusion
 MATERIALS AND METHODS
 REFERENCES
 

  1. Chen S 1998 Aromatase and breast cancer. Front Biosci 3:d922–d933
  2. Bajetta E, Zilembo N, Bichisao E, Martinetti A, Buzzoni R, Pozzi P, Bidoli P, Ferrari L, Celio L 2000 Tumor response and estrogen suppression in breast cancer patients treated with aromatase inhibitors. Ann Oncol 11:1017–1022[Abstract/Free Full Text]
  3. Plourde PV, Dyroff M, Dowsett M, Demers L, Yates R, Webster A 1995 ARIMIDEX: a new oral, once-a-day aromatase inhibitor. J Steroid Biochem Mol Biol 53:175–179[CrossRef][Medline]
  4. Smith IE, Dowsett M 2003 Aromatase inhibitors in breast cancer. N Engl J Med 348:2431–2442[Free Full Text]
  5. Baum M, Budzar AU, Cuzick J, Forbes J, Houghton JH, Klijn JG, Sahmoud T 2002 Anastrozole alone or in combination with tamoxifen versus tamoxifen alone for adjuvant treatment of postmenopausal women with early breast cancer: first results of the ATAC randomised trial. Lancet 359:2131–2139[CrossRef][Medline]
  6. Poulos TL, Finzel BC, Gunsalus IC, Wagner GC, Kraut J 1985 The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450. J Biol Chem 260:16122–16130[Abstract/Free Full Text]
  7. Zhou DJ, Korzekwa KR, Poulos T, Chen SA 1992 A site-directed mutagenesis study of human placental aromatase. J Biol Chem 267:762–768[Abstract/Free Full Text]
  8. Zhou D, Cam LL, Laughton CA, Korzekwa KR, Chen S 1994 Mutagenesis study at a postulated hydrophobic region near the active site of aromatase cytochrome P450. J Biol Chem 269:19501–19508[Abstract/Free Full Text]
  9. Graham-Lorence S, Amarneh B, White RE, Peterson JA, Simpson ER 1995 A three-dimensional model of aromatase cytochrome P450. Protein Sci 4:1065–1080[Medline]
  10. Auvray P, Nativelle C, Bureau R, Dallemagne P, Seralini GE, Sourdaine P 2002 Study of substrate specificity of human aromatase by site directed mutagenesis. Eur J Biochem 269:1393–1405[Medline]
  11. Laughton CA, Zvelebil MJ, Neidle S 1993 A detailed molecular model for human aromatase. J Steroid Biochem Mol Biol 44:399–407[CrossRef][Medline]
  12. Koymans LM, Moereels H, Vanden Bossche H 1995 A molecular model for the interaction between vorozole and other non-steroidal inhibitors and human cytochrome P450 19 (P450 aromatase). J Steroid Biochem Mol Biol 53:191–197[CrossRef][Medline]
  13. Kao YC, Cam LL, Laughton CA, Zhou D, Chen S 1996 Binding characteristics of seven inhibitors of human aromatase: a site-directed mutagenesis study. Cancer Res 56:3451–3460[Abstract/Free Full Text]
  14. Chen S, Zhang F, Sherman MA, Kijima I, Cho M, Yuan YC, Toma Y, Osawa Y, Zhou D, Eng ET 2003 Structure-function studies of aromatase and its inhibitors: a progress report. J Steroid Biochem Mol Biol 86:231–237[CrossRef][Medline]
  15. Williams PA, Cosme J, Sridhar V, Johnson EF, McRee DE 2000 Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity. Mol Cell 5:121–131[CrossRef][Medline]
  16. Williams PA, Cosme J, Ward A, Angove HC, Matak Vinkovic D, Jhoti H 2003 Crystal structure of human cytochrome P450 2C9 with bound warfarin. Nature 424:464–468[CrossRef][Medline]
  17. Favia AD, Cavalli A, Masetti M, Carotti A, Recanatini M 2006 Three-dimensional model of the human aromatase enzyme and density functional parameterization of the iron-containing protoporphyrin IX for a molecular dynamics study of heme-cysteinato cytochromes. Proteins 62:1074–1087[CrossRef][Medline]
  18. Pompon D, Liu RY, Besman MJ, Wang PL, Shively JE, Chen S 1989 Expression of human placental aromatase in Saccharomyces cerevisiae. Mol Endocrinol 3:1477–1487[Abstract/Free Full Text]
  19. Zhang F, Zhou D, Kao YC, Ye J, Chen S 2002 Expression and purification of a recombinant form of human aromatase from Escherichia coli. Biochem Pharmacol 64:1317–1324[CrossRef][Medline]
  20. Kagawa N, Hori H, Waterman MR, Yoshioka S 2004 Characterization of stable human aromatase expressed in E. coli. Steroids 69:235–243[CrossRef][Medline]
  21. Amarneh B, Simpson ER 1995 Expression of a recombinant derivative of human aromatase P450 in insect cells utilizing the baculovirus vector system. Mol Cell Endocrinol 109:R1–R5
  22. Jo Corbin C, Mapes SM, Lee YM, Conley AJ 2003 Structural and functional differences among purified recombinant mammalian aromatases: glycosylation, N-terminal sequence and kinetic analysis of human, bovine and the porcine placental and gonadal isozymes. Mol Cell Endocrinol 206:147–157[CrossRef][Medline]
  23. Cosme J, Johnson EF 2000 Engineering microsomal cytochrome P450 2C5 to be a soluble, monomeric enzyme. Mutations that alter aggregation, phospholipid dependence of catalysis, and membrane binding. J Biol Chem 275:2545–2553[Abstract/Free Full Text]
  24. Schoch GA, Yano JK, Wester MR, Griffin KJ, Stout CD, Johnson EF 2004 Structure of human microsomal cytochrome P450 2C8. Evidence for a peripheral fatty acid binding site. J Biol Chem 279:9497–9503[Abstract/Free Full Text]
  25. Hagerman DD 1987 Human placenta estrogen synthetase (aromatase) purified by affinity chromatography. J Biol Chem 262:2398–2400[Abstract/Free Full Text]
  26. Kellis JT, Jr., Vickery LE 1987 Purification and characterization of human placental aromatase cytochrome P-450. J Biol Chem 262:4413–4420[Abstract/Free Full Text]
  27. Yoshida N, Osawa Y 1991 Purification of human placental aromatase cytochrome P-450 with monoclonal antibody and its characterization. Biochemistry 30:3003–3010[CrossRef][Medline]
  28. Brueggemeier RW, Hackett JC, Diaz-Cruz ES 2005 Aromatase inhibitors in the treatment of breast cancer. Endocr Rev 26:331–345[Abstract/Free Full Text]
  29. Gonzalez A, Piferrer F 2002 Characterization of aromatase activity in the sea bass: effects of temperature and different catalytic properties of brain and ovarian homogenates and microsomes. J Exp Zool 293:500–510[CrossRef][Medline]
  30. Poulos TL 2003 Cytochrome P450 flexibility. Proc Natl Acad Sci USA 100:13121–13122[Free Full Text]
  31. Werck-Reichhart D, Feyereisen R 2000 Cytochromes P450: a success story. Genome Biol 1:REVIEWS3003
  32. Groves JT, Watanabe YJ 1988 Reactive iron porphyrin derivatives related to the catalytic cycles of cytochrome P-450 and peroxidase. Studies of the mechanism of oxygen activation. J Am Chem Soc 110:8443–8452[CrossRef]
  33. Groves JT, McMurry TJ 1985 Rev Port Quim 27:102–103
  34. Morand P, Williamson DG, Layne DS, Lompa-Krzymien L, Salvador J 1975 Conversion of an androgen epoxide into 17ß-estradiol by human placental microsomes. Biochemistry 14:635–638[CrossRef][Medline]
  35. Akhtar M, Calder MR, Corina DL, Wright JN 1982 Mechanistic studies on C-19 demethylation in oestrogen biosynthesis. Biochem J 201:569–580[Medline]
  36. Covey DF, Hood WF, Beusen DD, Carrell HL 1984 Hydroperoxides as inactivators of aromatase: 10ß-hydroperoxy-4-estrene-3,17-dione, crystal structure and inactivation characteristics. Biochemistry 23:5398–5406[CrossRef][Medline]
  37. Hahn EF, Fishman J 1984 Immunological probe of estrogen biosynthesis. Evidence for the 2ß-hydroxylative pathway in aromatization of androgens. J Biol Chem 259:1689–1694[Abstract/Free Full Text]
  38. Cole PA, Bean JM, Robinson CH 1990 Conversion of a 3-desoxysteroid to 3-desoxyestrogen by human placental aromatase. Proc Natl Acad Sci USA 87:2999–3003[Abstract/Free Full Text]
  39. Kao YC, Korzekwa KR, Laughton CA, Chen S 2001 Evaluation of the mechanism of aromatase cytochrome P450. A site-directed mutagenesis study. Eur J Biochem 268:243–251[Medline]
  40. Hackett JC, Brueggemeier RW, Hadad CM 2005 The final catalytic step of cytochrome p450 aromatase: a density functional theory study. J Am Chem Soc 127:5224–5237[CrossRef][Medline]
  41. Garrett WM, Hoover DJ, Shackleton CH, Anderson LD 1991 Androgen metabolism by porcine granulosa cells during the process of luteinization in vitro: identification of 19-oic-androstenedione as a major metabolite and possible precursor for the formation of C18 neutral steroids. Endocrinology 129:2941–2950[Abstract/Free Full Text]
  42. Giudici D, Ornati G, Briatico G, Buzzetti F, Lombardi P, di Salle E 1988 6-Methylenandrosta-1,4-diene-3,17-dione (FCE 24304): a new irreversible aromatase inhibitor. J Steroid Biochem 30:391–394[CrossRef][Medline]
  43. Covey DF, Hood WF 1981 Enzyme-generated intermediates derived from 4-androstene-3,6,17-trione and 1,4,6-androstatriene-3,17-dione cause a time-dependent decrease in human placental aromatase activity. Endocrinology 108:1597–1599[Abstract/Free Full Text]
  44. Numazawa M, Midzuhashi K, Nagaoka M 1994 Metabolic aspects of the 1ß-proton and the 19-methyl group of androst-4-ene-3,6,17-trione during aromatization by placental microsomes and inactivation of aromatase. Biochem Pharmacol 47:717–726[CrossRef][Medline]
  45. Graham-Lorence S, Khalil MW, Lorence MC, Mendelson CR, Simpson ER 1991 Structure-function relationships of human aromatase cytochrome P-450 using molecular modeling and site-directed mutagenesis. J Biol Chem 266:11939–11946[Abstract/Free Full Text]
  46. Chen S, Kao YC, Laughton CA 1997 Binding characteristics of aromatase inhibitors and phytoestrogens to human aromatase. J Steroid Biochem Mol Biol 61:107–115[CrossRef][Medline]
  47. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  48. Zhou DJ, Pompon D, Chen SA 1990 Stable expression of human aromatase complementary DNA in mammalian cells: a useful system for aromatase inhibitor screening. Cancer Res 50:6949–6954[Abstract/Free Full Text]



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