Structural Determinants of Aromatase Cytochrome P450 Inhibition in Substrate Recognition Site-1

Alan Conley, Samantha Mapes, C. Jo Corbin, Douglas Greger1 and Sandra Graham2

Department of Population Health and Reproduction, University of California School of Veterinary Medicine (A.C., S.M., C.J.C., D.G.), Davis, California 95616; and Department of Biochemistry, University of Texas Southwestern Medical Center (S.G.), Dallas, Texas 75235

Address all correspondence and requests for reprints to: Dr. Alan Conley, Department of Population Health and Reproduction, University of California School of Veterinary Medicine, Davis, California 95616. E-mail: ajconley{at}ucdavis.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The porcine gonadal form of aromatase cytochrome P450 (P450arom) exhibits higher sensitivity to inhibition by the imidazole, etomidate, than the placental isozyme. The residue(s) responsible for this functional difference was mapped using chimeragenesis and point mutation analysis of the placental isozyme, and the kinetic analysis was conducted on native and mutant enzymes after overexpression in insect cells. The etomidate sensitivity of the placental isozyme was markedly increased by substitution of the predicted substrate recognition site-1 (SRS-1) and essentially reproduced that of the gonadal isozyme by substitution of SRS-1 and the predicted B helix. A single isoleucine (I) to methionine (M) substitution at position 133 of the placental isozyme (I133M) was proven to be the critical residue within SRS-1. Residue 133 is located in the B'-C loop and has been shown to be equally important in other steroid-metabolizing P450s. Single point mutations (including residues 110, 114, 120, 128, 137, and combinations thereof among others) and mutation of the entire B and C helixes were without marked effect on etomidate inhibitory sensitivity. The same mutation (I133M) introduced into human P450arom also markedly increased etomidate sensitivity. Mutation of Ile133 to either alanine (I133A) or tyrosine (I133Y) decreased apparent enzyme activity, but the I133A mutant was sensitive to etomidate inhibition, suggesting that it is Ile133 that decreases etomidate binding rather than Met133 increasing enzyme sensitivity. Androstenedione turnover and affinity were similar for the I133M mutant and the native placental isozyme. These data suggest that Ile133 is a contact residue in SRS-1 of P450arom, emphasize the functional conservation that exists in SRS-1 of a number of steroid-hydroxylating P450 enzymes, and suggest that substrate and inhibitor binding are dependent on different contact points to varying degrees.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGENS ARE synthesized from androgens by the enzyme known as aromatase cytochrome P450 (P450arom). This enzyme is expressed in a number of tissues in the body, including male and female gonads, brain, placenta, and adipose tissue among others (1, 2). Expression is regulated in a distinct tissue-specific fashion and is critical to normal sexual development and subsequent fertility (3). However, it is becoming increasingly clear that either the lack of expression or overexpression of P450arom can lead to disease in a wide variety of tissues (4). Breast cancer is believed to develop and progress as a result of the local production of estrogen from the aberrant expression of P450arom in adipose stromal cells (5). As a consequence, great effort has been expended in devising pharmacological inhibitors of P450arom (6, 7, 8, 9), and some of the most recently developed, the so-called third generation imidazole and steroidal inhibitors, rank among the most potent and specific known for any P450. Early results with these compounds have been so encouraging that prophylactic treatment has been considered for women at high risk (10). Aberrant expression of P450arom has also been implicated in other diseases, such as endometriosis (11), and aromatase inhibitors have been used successfully for this (12) as well as a variety of other conditions (13, 14, 15). The potency of third generation aromatase inhibitors coupled with the possibility that they may be used more widely and that therapies may be prolonged has lead to concern over undesirable side-effects (16).

Structural models of P450s offer the promise of explaining and predicting substrate preferences and inhibitor specificities (17) as well as enabling the design of drugs that effectively and specifically modulate P450 function with minimal side-effects (18). The solved structures of several bacterial P450s form the backbone of most homology models (19) because microsomal P450s have been particularly difficult to crystallize. Mutagenesis has been an essential tool in testing and refining these to represent mammalian P450 isozymes (20), but it is still unclear what basic structural differences dictate the catalytic differences among them, substrate preferences and stereospecificity for instance. Two basic approaches have been taken in designing mutations. One identifies and targets residues based on the model itself, and generally a loss of function upon mutation is taken as evidence of the accuracy of the model. This has been the strategy used in almost all structure-function studies of P450arom (21, 22, 23, 24, 25, 26, 27, 28). These studies have relied on expression by transient transfection or in stably transfected cell lines in part because of difficulties in overexpressing P450arom in bacteria, which has been successful only after deletion of amino-terminal residues (29, 30). An alternative approach relies on catalytic or functional differences between closely related isozymes, from different species or within a species (31, 32). Interchanging nonidentical residues between different isozymes and thereby mapping function empirically has been used extensively and with notable success to gain significant insights into the substrate binding pocket of a number of different P450s from the 2A, 3A, 2B, and 2C families among others (20). Gotoh (33) used a computer analysis of the CYP2C family of P450s to identify six major domains, which he designated substrate recognition sites (SRS). These, he postulated, were involved in substrate binding and accounted for differences in substrate specificities between isozymes within the family. The results of chimeric enzyme and site-directed mutagenesis studies of SRSs in 3A4, 2B, and other P450 isozymes (34, 35, 36, 37, 38, 39) generally support this interpretation, although not precluding the likely importance of residues outside these regions (40, 41). To our knowledge, this approach, using catalytic differences among isozymes to design mutants, has never been used in structure-function studies of P450arom despite the fact that over a dozen mammalian P450arom enzymes have been cloned (2). The apparent functional conservation among aromatases from different species, even from different vertebrate classes, has left little in the way of catalytic differences that could be usefully mapped. However, the existence of multiple forms of porcine aromatase that have evolved within this species suggests a greater likelihood of functional divergence (42, 43). These might be more useful in structure-function studies and in the refinement of aromatase models.

Our laboratory was the first to recognize the existence of multiple isozymes of P450arom in the pig (42) that are encoded by separate genes located in a cluster on chromosome 1 (44, 45, 46), but are expressed in a strictly tissue-specific manner in gonads, placenta, and preattachment blastocysts (44, 47, 48). It remains the only known example among mammals. Functional studies of two of the porcine P450arom enzymes, the gonadal and placental isozymes, demonstrated that they exhibited differential sensitivity to the imidazole, etomidate. Specifically, the gonadal P450arom was sensitive to inhibition by etomidate, but the placental isozyme was not (42, 49). In recent studies estimated etomidate IC50 values for the two isozymes differed by 185-fold, but those for fadrazole, a well studied P450arom inhibitor, differed by only 2-fold. Furthermore, we constructed chimeric enzymes using the porcine gonadal, placental, and human aromatases (32), demonstrating them to be active, and mapping sensitivity to etomidate inhibition to the first 150 N-terminal amino acid residues of gonadal P450arom. Based on these data, and in the belief that etomidate might therefore provide a useful tool for structure-function analysis, we initiated further chimeric and site-directed mutagenesis experiments within this region of the placental isozyme to ascertain which specific residue(s) or structural motifs of the porcine gonadal P450arom were determinants of the unusual selectivity of etomidate inhibition. Since P450arom expression cannot be properly quantified in transfected cells, the consequences of mutagenesis on substrate utilization were further evaluated by kinetic analysis of recombinant enzymes over-expressed in insect cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The results of previous studies (32) mapped etomidate inhibitory sensitivity to the N-terminal third of the gonadal P450arom (residues 1–150). A conserved EcoRI restriction site in the gonadal and placental P450arom cDNAs was used in the current experiments to construct chimeric enzymes in which the first 50 N-terminal amino acids were switched (G50P and P50G) between the native enzymes (Fig. 1Go). The etomidate sensitivity of the resulting chimeric enzymes did not map to the 50 N-terminal residues (Table 1Go). However, inserting amino acid residues 101–150 from the gonadal to the placental isozyme (PG101–150P) was associated with a markedly decreased IC50 (increased sensitivity) of the resulting chimeric enzyme to etomidate inhibition to a level similar to that of the native gonadal P450arom. The substitution of these 50 residues of the placental isozyme was also accompanied by an apparent decrease in activity to gonadal levels (Table 1Go).



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Figure 1. Amino Acid Alignment of the N Terminus of the Porcine Placental, Porcine Gonadal, and Human P450arom to Residue 204

Shown are regions of identity (shaded areas) and sequence variance (*) in regions correlating roughly with the predicted exon boundaries. The EcoRI and BamHI restriction sites that were used in chimeric enzyme construction are also shown, along with the predicted membrane-spanning region, helixes (A, B, B', C, D, and E) and SRS-1, as defined by Gotoh (33 ).

 

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Table 1. Summary of Activity and Sensitivity to Etomidate Inhibition (IC50) of Native Porcine Placental (Pnative) and Gonadal (Gnative) Isozymes of P450arom and Chimeric Enzymes Generated from Them

 
Residues in the region between amino acids 101–150 of the placental P450arom were subsequently examined by site-directed mutagenesis. Substitution of functional motifs were targeted, namely those of the predicted B and C helixes and the region between them, identified as SRS-1 by sequence alignment (33) and encompassing the predicted B' helix, as depicted (Fig. 1Go). The sensitivities of the resulting mutant enzymes to inhibition by etomidate were assessed, as shown in Fig. 2Go, and IC50 values were estimated (Table 2Go). The mutant placental enzyme expressing the gonadal SRS-1 exhibited a markedly lower IC50 to etomidate, as did the mutant with the gonadal SRS-1 in combination with both the gonadal B and C helixes (Table 2Go). Mutants expressing the gonadal B and C helixes alone or in combination were not markedly different from the native placental P450arom in terms of etomidate inhibitory sensitivity, although there appeared to be a slight shift toward increased sensitivity of the B helix mutant. Nor was the combination of the gonadal B and C helixes effective in significantly changing the IC50 from that of the native placental enzyme. However, the B plus SRS-1 mutant exhibited an etomidate sensitivity indistinguishable from that of the native gonadal P450arom (Table 2Go and Fig. 2Go). The activity of the mutant enzymes was also seen to decrease in all constructs except those in which the B and C helixes alone were mutated. The SRS-1 and SRS-1/B helix mutants exhibited activities similar to that of the native gonadal isozyme, whereas the SRS-1/C and B/C mutants exhibited activities intermediate between those of the native placenta and gonadal isozymes. In general, it appeared that among mutants in which the B helix, SRS-1, and C helix were mutated, those with the highest activities were the most resistant to etomidate inhibition.



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Figure 2. Concentration-Dependent Inhibition of Native Porcine Placental and Gonadal Isozymes of P450arom, and Mutants of the Placental Isozyme by Etomidate

Constructs were generated by multiple point mutation within the regions of the predicted B and C helixes or SRS-1, as detailed in Materials and Methods. Native gonadal (Gnative), placental (Pnative), and mutant enzymes were subcloned into the cytomegalovirus promotor expression plasmid and transfected into human embryonal kidney 293 cells. Aromatase activity was determined by tritiated water release from [1ß-3H]androstenedione (also in Materials and Methods) in the presence of increasing concentrations of etomidate, shown as the log (nanomolar concentrations). The activities of all constructs are listed in Table 2Go, and inhibition was expressed as a percentage of the vehicle control (% control activity). Shown are the averages of at least three independent transfection experiments, each performed in duplicate. Note that placental mutants with gonadal B, C, and B plus C helixes have similar etomidate sensitivities to the native placental isozyme, whereas those with gonadal SRS-1, B plus SRS-1, and C plus SRS-1 exhibit gonadal-like sensitivity.

 

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Table 2. Summary of Activity and Sensitivity to Etomidate Inhibition (IC50), of Native Porcine Placental (Pnative) and Gonadal (Gnative) Isozymes of P450arom and Mutant Enzymes Generated from the Placental Isozyme

 
Analysis of point mutants within SRS-1 and outside this region (data from additional enzymes with point mutations in the B and C helixes not shown) determined that a substantial shift in etomidate sensitivity could be induced in the placental isozyme by the single point mutation in SRS-1, isoleucine-133 to methionine (I133M; Fig. 3Go). Although the IC50 of the native gonadal isozyme was still one third that of I133M, no other single amino acid change altered etomidate sensitivity to any comparable degree (Table 3Go). Additional experiments examined the effect of mutating residue 133 of the placental isozyme to an uncharged polar, but aromatic, amino acid (tyrosine, I133Y), or to a smaller, but still aliphatic, side-chain (alanine, I133A), as well as to introduce the Met133 mutation into an entirely different context, that of the human P450arom (H-I133M). The porcine placental I133A mutant exhibited an increased sensitivity to etomidate (25-fold difference in IC50 compared with the native placental P450arom), but the activity of the I133Y mutant was too low to enable an evaluation of etomidate sensitivity (Table 4Go). Finally, introducing the I133M mutation in the human P450arom (H-I133M) induced a similar marked increase in etomidate sensitivity to that seen with the same porcine placental mutant (Table 4Go). The native human P450arom had an IC50 of 121 ± 21 µM, and the single amino acid change, H-I133M, reduced the IC50 to 6.4 ± 1.5 µM, effectively increasing the sensitivity of the enzyme to etomidate inhibition almost 20-fold (Table 4Go).



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Figure 3. Concentration-Dependent Inhibition of Native Porcine Placental and Gonadal Isozymes of P450arom, and Mutants of the Placental Isozyme by Etomidate

Constructs were generated by point mutation within and around the predicted SRS-1, as detailed in Materials and Methods. Native gonadal (Gnative), placental (Pnative), and mutant enzymes were subcloned into the cytomegalovirus promoter expression plasmid and transfected into human embryonal kidney 293 cells. Aromatase activity was determined by tritiated water release from [1ß-3H]androstenedione (also in Materials and Methods) in the presence of increasing concentrations of etomidate, shown as the log (nanomolar concentrations). The activities of all constructs are listed in Table 3Go, and inhibition of activity was expressed as a percentage of the vehicle control (% control activity). Shown are the averages of at least three independent transfection experiments, each performed in duplicate. Note that only the placental point mutant I133M exhibited a shift in etomidate sensitivity from the native placental isozyme.

 

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Table 3. Summary of Activity and Sensitivity to Etomidate Inhibition (IC50) of Native Porcine Placental (Pnative) and Gonadal (Gnative) Isozymes of P450arom and Mutant Enzymes Generated from the Placental Isozyme

 

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Table 4. Summary of Activity and Sensitivity to Etomidate Inhibition (IC50) of Native Porcine Placental (Pnative), Human (Hnative), and Porcine Gonadal (Gnative) Isozymes of P450arom and Mutant Enzymes

 
The results of the experiments with these mutant enzymes also suggested that catalytic activity and sensitivity to etomidate were not necessarily correlated. For instance, the lowest activity among the mutants (Table 3Go) was exhibited by the SRS-1 mutant P450arom, P120L, that had one of the highest observed IC50 values. However, immunoblot analysis of all expressed mutant enzymes indicated that there was certainly variability in the efficiency of expression among constructs (Fig. 4Go), and that this may have accounted for the apparently decreased activity of some. The native gonadal P450arom was the most obvious example (Fig. 4AGo), wherein the expression of this construct appeared consistently to be low compared with that of most others. Although immunodetectable protein need not necessarily represent active P450, it appeared in other cases that a decrease in activity was probably not due to a failure of expression, suggesting that the catalytic activity of enzymes derived from certain constructs was truly low. Specifically, the low activity of the SRS-1 and B helix plus SRS-1 compared with the B helix, C helix, or B plus C helix mutants is most likely to reflect altered metabolic function (Fig. 4AGo). Similarly, the low activity of P120L compared with S114C or Y128H mutants appeared unlikely to have resulted from lowered expression (Fig. 4BGo).



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Figure 4. Western Immunoblot Analysis to Evaluate the Level of Expression of P450arom in Human HK293 Cells

Cells were transfected with expression constructs encoding for native placental and gonadal forms of porcine P450arom and several mutants. Cells were scraped from wells after the completion of activity analyses and pooled across replicates, and crude cell lysates (50 µg/lane) were used for immunoblot analysis as detailed in Materials and Methods.

 
The apparent differences in enzyme activities of the native isozymes and the placental I133M mutant, suggested by transient transfection studies, were further investigated using recombinant enzymes. These were overexpressed in insect cells using baculovirus vectors, quantified by difference spectroscopy, and supported by NADPH-cytochrome P450 reductase (reductase) in reconstituted assays. All enzymes appeared saturated or near saturated at 300 nM androstenedione, and estimates of substrate affinity were calculated from double reciprocal plots (Fig. 5Go and Table 5Go). The native placental and mutant I133M enzymes had similar substrate affinities, but the apparent Km of the gonadal P450arom for androstenedione was considerably lower. It was also clear from these plots that there was evidence of inhibition of aromatization at high androstenedione concentrations, particularly for the gonadal isozyme (Fig. 5Go). Therefore, saturation analysis with reductase was conducted with androstenedione at 433 nM, and the calculated estimates of turnover numbers were made from double reciprocal plots of these data. As reductase affinities did not differ (data not shown), saturation curves have been presented to allow easier comparison among turnover rates (Fig. 6Go). The placental enzyme turned over substrate at a consistently higher rate than either the I133M mutant or the gonadal P450arom (Fig. 6Go). Overall, the native placental isozyme was 5 times faster than the gonadal P450arom, and the mutant I133M was 4 times faster than the gonadal isozyme (Table 5Go). Thus, although the I133M mutation of the placental isozyme radically altered its sensitivity to etomidate inhibition, there was relatively little effect on catalytic efficiency toward the substrate, androstenedione.



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Figure 5. Double Reciprocal Plots of Substrate (Androstenedione, Nanomolar Concentrations) vs. Velocity (V; Minutes-1) for Reconstituted Activities of the Native and Mutant Porcine P450arom Enzymes

cDNAs encoding the native and mutant enzymes were subcloned into bacmid and transposed to generate recombinant baculovirus vectors, which were used subsequently to express and prepare recombinant enzymes from insect cells, as described in Materials and Methods. Aromatase activity was reconstituted using 2 nM gonadal (Gnative), placental (Pnative), or mutant (I133M) placental P450arom together with 0.1–3.3 mM androstenedione, 150 nM recombinant NADPH-cytochrome P450 reductase (reductase), and an NADPH-generating system and was measured by tritiated water release from [1ß-3H]androstenedione. Plots were generated by linear regression analysis from three independent experiments.

 

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Table 5. Kinetic Properties of Recombinant Native Placental (Pnative), Gonadal (Gnative), and the I133M Mutant Placental Isozyme of Porcine Aromatase Cytochrome P450

 


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Figure 6. Saturation Curves of Recombinant Porcine Native and Mutant P450arom Enzymes Reconstituted with Recombinant NADPH-Cytochrome P450 Reductase (Reductase)

cDNAs encoding the native and mutant enzymes were subcloned into bacmid and transposed to generate recombinant baculovirus vectors, which were used subsequently to express and prepare recombinant enzymes from insect cells, as described in Materials and Methods. Aromatase activity was reconstituted using 2 nM gonadal (Gnative), placental (Pnative), or mutant (I133M) placental P450arom together with reductase (0–40 nM) and androstenedione (433 nM) and an NADPH-generating system. Activity was measured by tritiated water release from [1ß-3H]androstenedione in three independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Structure-function studies and molecular modeling of P450s have been driven not only by a desire to understand differences in substrate specificity, but also in part by the need to design inhibitors with the potential to selectively inactive specific isozymes (18). The molecular basis of P450 substrate specificity remains a mystery. Moreover, it is equally unclear exactly how steroidal or nonsteroidal inhibitors occupy the active site (50) and whether their binding is reliant on the same SRSs or involves other contact residues (27). As ligands of P450s, steroid substrates induce spectra (type I) with very different maxima and minima than do imidazoles (type II), for instance, indicative of shifts in the heme-ligand interaction within the pocket (51, 52). Etomidate does induce a type II spectrum, and this can be reversed to a type I by increasing concentrations of androstenedione, providing direct evidence of competition between substrate and inhibitor for binding in the active site (data not shown). It seems probable that molecules assume varying orientations relative to specific contact residues within the active site, if only based on the distinct spectral characteristics of the P450 bound with substrate vs. inhibitor. The data presented here provide direct and convincing support for this view in identifying, by mutation analysis, a single residue critical in the binding of an imidazole to human and porcine aromatases, and in further showing that this same mutation did not significantly affect substrate utilization. This suggests that allelic variants or mutations of P450arom that may be silent relative to estrogen synthesis may nonetheless exhibit very different sensitivities to pharmacological or perhaps even diet-derived aromatase inhibitors. Flavonoids consumed with vegetables and other plants or as dietary supplements are a case in point (53, 54). Not only are these compounds capable of inhibiting P450arom, but their inhibitory potency toward human P450arom has been shown to be affected by mutation of the same residue (Ile133) shown here to determine etomidate binding affinity (55). We conclude that there are clear differences in the degree of involvement of certain contact points within the active site relative to the binding of substrates and potential P450 inhibitors. Such differences may prove valuable in the design of therapeutics with tissue-specific effects, perhaps analogs of etomidate itself or even naturally occurring flavonoids, whose potency might be differentially modified by local substrate concentrations based on competitive binding. Equally, the definition of critical active site residues and the effects of specific mutations may prove useful in predicting the response of individuals to treatments, dietary supplements, or even inhibitors in the diet from genotypic screens.

This is the first study to examine aromatase function after overexpression of native and mutant enzymes at sufficient levels to allow quantification by difference spectroscopy. Consequently, the kinetic analysis conducted using recombinant protein to reconstitute enzyme activities provided the first truly quantitative estimates of the effects of point mutation on P450arom turnover number (apparent maximum velocity) as well as apparent affinity. Previous studies of P450arom using transient or stable transfection examining the importance of specific residues, predicted to be involved in substrate recognition, as determinants of inhibitor binding (17, 26, 27, 55) have been unable to estimate the levels of active P450 expression. However, our study is not the first to examine the effect of mutation of Ile133, which has been investigated relative to the potency of a number of P450arom inhibitors used clinically. Chen and colleagues (17, 55) substituted Ile133 of human P450arom with tyrosine (and tryptophan), using expression in transfected CHO cells. These mutations had no effect on IC50 values of the several clinical inhibitors studied, but apparently decreased enzyme activity. Similarly, we found no difference between the gonadal and placental P450arom isozymes in their sensitivities to the imidazole inhibitor, fadrazole (32). These data suggest that Ile133, although critical for etomidate, may not be important in the binding of other imidazole inhibitors in the P450arom active site. Again, due to low levels of enzyme generated in the expression systems used, none of these prior investigations was able to precisely quantify the effects of mutation on substrate turnover, and even substrate affinities varied depending on whether analyses were conducted in whole cells or microsomes (27).

We believe that the current study is also the first to use known differences in inhibitor selectivity for a specific isozyme of aromatase to map the amino acid(s) that imparts this property. This strategy has been used successfully to map determinants of inhibitor sensitivity of 2B and several other P450 isozymes with interesting results. In several cases, determinants of inhibitor sensitivity involved not only the region defined by Gotoh (33) as SRS-1, but residues within in it that, as shown by alignment (Fig. 7Go), correspond to Ile133 of the porcine placental P450arom shown here to be the critical determinant of etomidate inhibition. Halpert and He (56) demonstrated that valine at position 114 (Val114) of P450 2B1 rendered the enzyme susceptible to inactivation by chloramphenicol, but alanine (Ala114) did not. Most recently, isoleucine 114 of P450 2B4 was also shown to be a critical residue in inhibition by 4-phenylimidazole (57). These data are of interest for two reasons. First, there are structural similarities between the phenylimidazoles and etomidate; both are small molecules with an aromatic side group. In this regard it is interesting to note that the presence of isoleucine at position 114 of P450 2B4 was associated with an increase in susceptibility to inhibition, whereas an increase in inhibitor sensitivity of porcine placental P450arom to etomidate was seen after the replacement of Ile133 with methionine. Secondly, mutations at position 114 altered substrate utilization, the regio-selectivity of hydroxylation of androstenedione specifically (58). The above data highlight the contrasting findings reported here showing a marked effect of mutation of the homologous residue (I133M) on the susceptibility P450arom to etomidate inhibition without markedly affecting androstenedione metabolism.



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Figure 7. Peptide Sequence Alignment of the Predicted SRS-1 Regions of Porcine Native Placental (Pnative) and Gonadal (Gnative) P450arom with Rabbit Cytochromes P450 2B4, 2B5, and 2C5, and Rat Cytochrome P450 2B1

The residue number is shown at the left, and those residues shared among other P450s are boxed. Ile133 of the placental P450arom is marked by an asterisk, and its number is designated. Note the sequence conservation through the region surrounding placental P450arom Ile133.

 
Although androstenedione turnover was only minimally affected in the methionine (I133M) mutant compared with the native placental P450arom (5.4 ± 1.4 vs. 7.0 ± 0.3/min, respectively; Table 5Go), our data indicate that this residue is involved in substrate binding in the P450arom active site. Specifically, substitution of Ile133 to valine, alanine, or tyrosine appeared to decrease the aromatization of androstenedione. Although these observations are based only on the results of transient transfection studies and were conducted without kinetic analysis at a single substrate concentration, they are consistent with the results of Chen and colleagues (27), who reported a similar apparent loss of activity with the same tyrosine substitution (I133Y) of the human P450arom. Moreover, this appears to hold true for other P450s. In fact, despite the poorly shared identity and millions of years of evolution that have occurred since these enzymes diverged (59), this residue has remained a critical determinant of substrate specificity in several other microsomal P450s among the 2A, 2B, 2C, and 3A families (20, 60, 61). Within SRS-1 of several of these enzymes (Fig. 7Go), Ile133 of P450arom is analogous to residues 117 of the mouse P450 2a5, 114 of rat 2B1, 114 of 2B4, 113 of 2C4 and 2C5, and 119 of 3A4. Mutagenesis studies in each case have identified these residues as being critically important in substrate binding (20, 60, 62). For mouse P450 2a5, alanine at position 117 promotes 7{alpha}-hydroxylation of dehydroepiandrosterone, whereas valine promotes 2{alpha}-hydroxylation (63). Substitution of the corresponding residue in P450 2B1 at position 114 from isoleucine to phenylalanine in combination with a change of leucine 58 to phenylalanine decreased 16ß-hydroxylation of androstenedione and testosterone without appreciably affecting 16{alpha}-hydroxylation or 17-keto formation (64). Studies of P450 2B4 demonstrated that mutating isoleucine 114 to phenylalanine 114 influenced the orientation of androstenedione binding so as to promote 16ß- rather than 16{alpha}-hydroxylation favored by the wild-type enzyme (58). Mutation of valine 113 of P450 2C4 to alanine, as it exists in the isozyme 2C5, significantly lowered the apparent Km of the enzyme for 21- hydroxylation of progesterone (65). Finally, four different mutations of serine 119 of P450 3A4 were shown to alter the ratios of 2ß- to 6ß-hydroxylation of both testosterone and progesterone (39). Despite the fact that the porcine P450arom isozymes share 20% or less amino acid identity with rat 2B1 and rabbit 2B4, 2B5, and 2C5, not much better than with the crystallized prokaryotic P450s, there appears to be much higher sequence conservation within the predicted SRS-1 region (Fig. 7Go) compared with other SRS regions, at least among the mammalian P450s. Therefore, it appears that SRS-1 is a highly conserved active site epitope and that the extensive structure-function studies previously conducted in a variety of microsomal P450s have great relevance to an understanding of P450arom.

Despite the weight of functional evidence from mutagenesis, in the absence of crystal data for an aromatase protein, the precise location of amino acid residue 133, and how etomidate actually inhibits enzyme activity, remain matters of speculation. However, given the weight of evidence supporting structural conservation of SRS-1, existing models of P450arom and those of other microsomal P450s may provide valuable insights. It may be equally pertinent in this regard that androgens or other steroids are often functionally relevant substrates for these other microsomal enzymes. Interestingly also, isoleucine appears in position 133 in all other mammalian aromatases whose sequence has been reported to date (2), even that of the third porcine blastocyst isozyme (48). In other words, with the exception of the porcine gonadal P450arom, this residue appears to be invariant in aromatases of all studied vertebrates. The analysis of 2C5 crystals, accomplished recently by Williams et al. (66), indicates that alanine 113, the residue corresponding to position 133 of the porcine and human P450arom, lies close to the heme and is actually in the active site (67). The data presented here and predictions from models of P450arom (17) are consistent with this view. It is important to note, however, that neither mutation of isoleucine 133 to methionine of the placental isozyme (I133M) nor mutation of SRS-1 itself entirely recapitulated the sensitivity to etomidate inhibition seen with the gonadal isozyme, which was seen only in combination with the mutation of the B helix. Thus, the current data are consistent with a possible interaction between SRS-1 and the B helix as an additional component influencing etomidate inhibitory potency. Possible interactions between SRS-1 and specific B helix residues together with a careful evaluation of mechanisms of inhibition will need to be examined more closely in future experiments. Regardless, the data presented emphasize the structural and functional similarities that exist in SRS-1 between P450arom and a number of other microsomal P450 enzymes. Based on our data, the new information provided on 2C5 is likely to be very relevant to P450arom structure as current models are revised.

Finally, in addition to the identification of the residue most critical to the binding of etomidate, this report represents the only assessment of recombinant aromatases isolated from insect cells to date that directly compares the functions of different P450arom proteins. The turnover number for the native porcine placental P450arom reported here accords well with those for recombinant (68, 69) and purified human P450arom (70) and with activity differences between isozymes after transient (42) or stable transfection, especially after saturation with reductase (71). Direct comparisons will have to be made to determine whether differences in catalytic efficiency exist between species, as they clearly do between the native porcine placental and gonadal isozymes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
All general chemicals were of highest quality, purchased from AMRESCO (Solon, OH), Fisher Scientific (Pittsburgh, PA), or Sigma (St. Louis, MO). Restriction enzymes and DNA-modifying enzymes were obtained from Promega Corp. (Madison, WI) or New England Biolabs, Inc. (Beverly, MA). Tissue culture media, antibiotics, trypsin, HEPES, and lipofectamine were obtained from Life Technologies, Inc. (Gaithersburg, MD).

Chimerigenesis and Site-Directed Mutagenesis
Full-length porcine placenta (P) and gonadal (G) aromatase cDNA were assembled in the cytomegalovirus promoter-5 (pCMV5) expression plasmid as described previously (32). Briefly, restriction enzyme digests of native G and native P with BamHI and ClaI were used to generate 500-bp middle cassette fragments that were exchanged to make G-P-G and P-G-P. Digestions of G-P-G and P-G-P used the internal ClaI coupled with StuI, a site in the vector, to switch out the C-terminal fragment and create G-P-P and P-G-G. Additional cassette switches were then made using EcoRI in exon III coupled with a restriction site in the vector (Fig. 1Go). This manipulation effectively switched the first 40 or so amino acids of each native enzyme. The same cassette switch applied to existing G-P-P and P-G-G chimerics to rebuild the N-terminal amino acid residues of those constructs, creating chimeric P and G enzymes, but with residues of exon IV from the other isozyme (PG101–150P and GP101–150G). Thus, these latter constructs tested the involvement of residues in the B, B', and C helixes and those in between. After restriction enzyme digests, all DNA was separated by agarose gel electrophoresis, purified using the QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA), and eluted in 30 µl H2O. Ligation of DNA fragments was carried out overnight at 15 C and subsequently used to transform DH5{alpha} competent cells (Life Technologies, Inc.). The correct sequences of all constructs were confirmed before use. The designation of point mutants was based on the sequence of the placental isozyme to facilitate comparisons with other mammalian aromatases that would otherwise be confused by the two missing residues at positions 10 and 11 of the gonadal P450arom. Site-directed mutagenesis of the cDNA encoding the porcine placental isozyme was performed using a QuikChange Site-Directed Mutagenesis Kit essentially as described by the manufacturer (Stratagene, La Jolla, CA). Primers, designed to be 32–42 bp in length, with a 35–51% G-C content and a melting temperature of 74–80 C, were purified by PAGE (Life Technologies, Inc.). Each PCR used 50 ng cDNA and 125 ng of each primer, reaction buffer (1x final concentration), deoxy-NTP mix (10 mM), and 2.5 U PfuTurbo DNA polymerase. Cycling parameters were 1 cycle at 95 C for 30 sec and 16 cycles of 95 C for 30 sec, 55 C for 2 min, and 68 C for 12 min. The PCR products were digested with DpnI for 1 h at 37 C, then used to transform Epicurian Coli XL1-Blue-competent cells. Colonies were isolated, and all resulting plasmids were completely sequenced to verify both the introduction of the mutation and the integrity of the rest of the insert.

Transient Transfection
Cells of the human 293 fetal kidney line were maintained in continuous monolayer culture at 37 C in 5% CO2 in DMEM, high glucose, 10% fetal bovine serum (Omega Scientific, Tarzana, CA), and 10 mM HEPES. For transfection, cells were plated in 12-well plates (Costar, Fisher Scientific) at a density of 2.5 x 105 cells in 1 ml medium/well. Twenty-four hours later, cells were incubated in the presence of plasmid DNA (0.6 µg/well) and lipofectamine reagent (1.3 µl/well) for 5 h in OptiMEM (Life Technologies, Inc.). After transfection, cells were placed in growth medium. Analyses of aromatase activity in the presence of varying concentrations of etomidate were conducted 48 h post transfection by tritiated water release, as detailed below. Levels of P450arom expression were also assessed in each culture by immunoblot analysis. At least three independent experiments were conducted in duplicate with each construct along with native placental and gonadal P450 aromatases as controls. The concentration-dependent inhibition of activity was plotted as a percentage of the vehicle control for both the mutant and native enzymes and expressed as the concentration of etomidate that suppressed aromatase activity by 50% (IC50).

Tritiated Water Assay
Aromatase activity was estimated by measuring the incorporation of tritium from [1ß-3H]androstenedione into 3H2O (NEN Life Science Products, Wilmington, DE). Incubations with androstenedione (10% labeled, 90% radioinert; Steraloids, Wilton, NH) were carried out at 37 C in the presence or absence of inhibitor. Etomidate (gift from Abbott Laboratories, North Chicago, IL) was added in ethanol (<1%) to reach final target concentrations (0.1–333 µM) in 0.5 ml OptiMEM or buffer. After incubation, reactions were stopped by addition of 0.5 ml cold 30% trichloroacetic acid. Samples were extracted subsequently with 1 ml chloroform. The aqueous phase (0.5 ml) was removed and combined with 0.5 ml 5% charcoal and 0.5% dextran suspension. After centrifugation for 30 min at 2000 x g, 0.5 ml was removed and quantified by liquid scintillation counting. Evidence verifying estrone product formation by HPLC and the close correlation between estrogen formation and tritium release has been presented in several previous reports from this laboratory (42, 43, 71).

Western Immunoblot Analysis
Cells were homogenized in PBS containing 1% sodium cholate and 0.1% SDS and sonicated for 3 sec. Equal amounts of protein from each replicate were first pooled, so as to represent the average expression for each construct and therefore mean activity, and then aliquots (50 µg) were subjected to SDS-PAGE (8% gel) in buffer containing 50 mM Tris, 383 mM glycine, 0.1% sodium dodecyl sulfate, and 0.4 mM EDTA. Separated proteins were transferred by electroblot onto polyvinylidene difluoride membranes, immunoblotted with antisera (1:2000) raised against recombinant human P450arom protein (courtesy of Dr. N. Harada, Fujita Health University, Aichi, Japan), and detected by chemiluminescence (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL) as described previously (43, 71).

Baculovirus Cloning, Overexpression, and Purification
Native porcine gonadal and placental P450arom and the mutant placental I133M mutant cDNAs with PCR-generated C-terminal His tags were subcloned into pFastBac1 (Life Technologies, Inc.). Transformation and transposition into bacmid DNA were carried out according to the manufacturer (Bac-to-Bac Baculovirus Expression System, Life Technologies, Inc.). PCR was used to identify recombinant clones, which were subsequently used to transfect mid-log phase Sf9 insect cells grown in SF900-II SFM (Life Technologies, Inc.). Viral titer was determined by plaque assay. Rounds of amplification, infecting Sf9 cells at a multiplicity of infection between 0.05 and 0.1, were repeated until the titers reached at least 1 x 107 pfu/ml. Protein overexpression was conducted in High Five insect cells (Invitrogen, Carlsbad, CA), grown in 50- or 100-ml suspension cultures in Express Five SFM (Life Technologies, Inc.), infected at a multiplicity of infection of at least 5, and grown for 72 h at 26 C in the presence of ferrous ammonium sulfate (0.2 mM) and {delta}- aminolevulinic acid (0.3 mM). Cell pellets were resuspended at a concentration factor of 20x in cold buffer [0.1 M KPO4 (pH 8.0), 20% glycerol, 5 mM ß-mercaptoethanol (BME),5 mM 3-[3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), and 0.5 mM phenylmethylsulfonylfluoride], homogenized in a tissue grinder, and briefly sonicated. Solubilization was continued by gentle rotation for 1 h at 4 C. The unsolubilized fraction was removed by centrifugation for 1 h (100,000 x g), and the supernatant was moved to a fresh tube, combined with 1 ml Ni-NTA agarose (QIAGEN, Valencia, CA), and incubated on a rotator for 1 h at 4 C. The suspension was then loaded into a 5-ml disposable column, and the resin was packed by gravity flow. The column was washed twice with 4 ml buffer [0.1 M KPO4 (pH 7.4), 20% glycerol, 5 mM BME, 5 mM CHAPS, and 20 mM imidazole], and the protein was eluted in a total volume of 2 ml wash buffer containing 200 mM imidazole. The eluate was dialyzed overnight against dialysis buffer [0.1 M KPO4 (pH 7.4), 20% glycerol, 5 mM BME, and 1 mM CHAPS]. P450arom content was determined by carbon monoxide difference spectrum. Spectra were not visible in the presence of imidazole and therefore were not detected until after dialysis.

Substrate Kinetics
Recombinant P450 reductase (gift from Dr. Ron Estabrook, University of Texas Southwestern Medical Center, Dallas, TX) was used to reconstitute activity with purified recombinant porcine native P450arom and the I133M mutant placental P450arom (2 nM) in a total assay volume of 0.5 ml. Substrate saturation was conducted with a reductase level of 150 nM and androstenedione concentrations from 10–3333 nM. For reductase saturation experiments, androstenedione concentrations were kept at 433 nM, and reductase concentrations were varied from 2–150 nM. All reactions were run for 30 min in the presence of an NADPH-generating system (1 mM NADPH, 2 mM NADP, 17 mM glucose-6-phosphate, and 1 U glucose-6-phosphate dehydrogenase) at 37 C. The reactions were stopped with 0.24 ml cold 30% trichloroacetic acid and assayed by tritiated water release.


    ACKNOWLEDGMENTS
 
The authors express their sincere thanks to Abbott Laboratories and Ciba-Geigy Corp. for supplying the P450 inhibitors used in the study.


    FOOTNOTES
 
This work was supported by Grant HD-36913 (to A.C.).

1 Current address: Templar Sciences, 1981 Pine Hall Road, State College, Pennsylvania 00000. Back

2 Paid from NIH Grants GM-43479 and GM-50858 to Julian A. Peterson. Back

Abbreviation: P450arom, Aromatase cytochrome P450.

Received for publication October 4, 2001. Accepted for publication February 25, 2002.


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