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E R Simpson

Publications and source records attributed to E R Simpson.

At least 73 records · Page 4Linked to original sources
PubMed1997

Regulation of aromatase expression in the ovary and placenta: a comparison between two species.

The conversion of C19 steroids to estrogens is catalysed by aromatase P450 (P450arom; product of the CYP19 gene). Tissue sites of expression include the gonads and brain; however, in a small subset of mammals, P450arom is also expressed in the placenta. In humans, gonadal expression employs a promoter proximal to the start site of translation, whereas expression in the placenta relies on a promoter which is distal to this site. We characterized the bovine CYP19 gene in both the ovary (OV) and placenta (PL), to determine if this method of regulation of tissue-specific expression is employed in other species. Both bovine and human species express P450arom in these tissues, however, the pattern of expression is very different. In both humans and cattle, P450arom is expressed in the granulosa cells of the ovary, however, after ovulation only the luteinized granulosa cells of the human continue to express P450arom. There is a high degree of sequence identity (>70%) shared between humans and cattle in the OV-specific 5'-flanking DNA, whereas little identity (<40%) was found between humans and cattle in the PL-specific DNA. Promoters and 5'-flanking regions of human and bovine CYP19 genes were subcloned into a luciferase (LUC) vector. Bovine PL-specific constructs transfected into JEG-3 human choriocarcinoma cells failed to express LUC activity. When OV-specific constructs were transfected into luteinized bovine granulosa cells, there was no change in LUC activity in cells transfected with the bovine constructs after treatment with forskolin, whereas all of the corresponding human constructs expressed LUC activity. The bovine 5'-flanking DNA lacks a cAMP-responsive element-like sequence (CLS) critical for cAMP-stimulated transcription of P450arom in the human ovary. With the absence of this CLS sequence in the bovine gene, there appears to be little enhancement of transcription by cAMP in the portion of the 5'-flanking region studied so far. When the analogous region of the bovine promoter was mutated to the human CLS, only a partial restoration of LUC activity was observed. An additional element therefore appears to be important in preventing the full expression of the bovine CYP19 gene in luteinized granulosa cells.

Animals↗
PubMed1996

Detection of aromatase cytochrome P450, 17 alpha-hydroxylase cytochrome P450 and NADPH:P450 reductase on the surface of cells in which they are expressed.

Using the membrane impermeant probe NHS-LC-biotin, we show in this report that a fraction of aromatase P450 (P450arom), the enzyme that catalyzes estrogen biosynthesis, is present at the surface of cells in which it is expressed, either endogenously or as a consequence of transfection. The same findings were obtained for a truncated form of P450arom lacking the putative membrane-spanning region, thus suggesting the presence of other membrane-spanning region(s) within its structure. P450arom is not unique in this regard as we find that a fraction of 17 alpha-hydroxylase P450 as well as NADPH:P450 reductase also are present at the cell surface. It is therefore possible that a number of microsomal P450s are expressed at the cell surface in this fashion.

Animals↗
PubMed1996

Expression of transcripts of interleukin-6 and related cytokines by human breast tumors, breast cancer cells, and adipose stromal cells.

The expression of transcripts of cytokines of the interleukin-6 (IL-6) family has been examined in human breast tumors, breast cancer cell lines, and adipose stromal cells, by means of reverse transcription polymerase chain reaction amplification. Of the six breast tumor samples examined, all expressed transcripts encoding IL-6 and Leukemia Inhibitory Factor (LIF). Four of the samples also expressed transcripts for oncostatin M (OSM) and IL-11, and three expressed the IL-6 receptor. Adipose stromal cells expressed IL-6, IL-11 and LIF, but not the IL-6 receptor, consistent with previous conclusions that IL-6 activity in these cells required addition of IL-6 soluble receptor. In the case of T47D cells, expression of IL-11 protein was confirmed by immunotitration. Moreover, in these cells, expression of IL-11 transcripts was induced 3-fold by addition of estradiol to the culture medium. These results add credence to our previous proposal that breast cancer development is regulated in part by local autocrine and paracrine mechanisms via epithelial/mesenchymal interactions, in which estrogen produced by stromal cells surrounding the tumor acts to stimulate the production of growth factors and cytokines by the tumor cells. Some of these may act to stimulate further the growth and development of the tumor, while these or other factors may act on the surrounding mesenchymal cells in a paracrine fashion to stimulate aromatase expression in the presence of glucocorticoids. Thus, a positive feedback loop is established which leads to the development and growth of the tumor.

Adipose Tissue↗
PubMed1996

17alpha-Hydroxylase gene expression in the bovine ovary: mechanisms regulating expression differ from those in adrenal cells.

17alpha-Hydroxylase cytochrome P450 (P450(17alpha)) is the enzyme which synthesizes C19 steroids in a two-step reaction in which 17alpha-OH pregnenolone is an intermediate. In the bovine and human adult female, 17alpha-hydroxylase is expressed in adrenocortical cells where 17alpha-OH pregnenolone and 17alpha-OH progesterone are precursors of cortisol, and in theca cells of the ovary where these intermediates are precursors of C19 steroids. In both adrenal cortex and theca, 17alpha-hydroxylase gene expression is stimulated by cyclic AMP (cAMP). The aim of this study was to determine the mechanism regulating 17alpha-hydroxylase gene expression in the bovine ovary. Our results indicate that the bovine 17alpha-hydroxylase gene is regulated in a tissue-specific fashion. Primer extension and S1 nuclease protection assays reveal that the start site of transcription in the theca is identical to that in the adrenal. Transfection studies employing beta-globin reporter gene constructs fused to successive deletions of the 5' regulatory region of the bovine 17alpha-hydroxylase gene indicate that sequences between -80 and -37 basepairs (bp) (CRS2) confer cAMP-regulated transcription in bovine theca cells in culture. These results are in contrast to similar studies conducted in bovine adrenocortical cells, which indicate that the major cAMP response element (referred to as CRS1) is located at -243 to -225 bp. The Ad4 element (AGGTCA, -42 to -37 bp) within CRS2, which has been shown to be involved in cAMP responsiveness in other steroidogenic P450 genes, cannot by itself confer cAMP-regulated reporter gene expression in bovine cells. These results indicate that in the cow, 17alpha-hydroxylase gene expression is regulated in a tissue-specific fashion, and that this regulation may be conferred, at least in part, by the use of tissue-specific cis-acting elements in the bovine 17alpha-hydroxylase gene.

Adrenal Cortex↗
PubMed1996

Functional aromatase expression in porcine adrenal gland and testis.

The expression of aromatase cytochrome P450 (P450arom) in the adrenal glands, testes, and placentas of fetal and newborn pigs was investigated. Western immunoblot analysis detected a single 48-50-kDa protein band in these tissues as well as in other porcine tissues known to express P450arom including Day 12 tubular conceptuses, theca interna, and granulosa. Slight differences in migration suggested that the P450arom protein expressed in the testis was larger than that in the adrenal gland, which was, in turn, larger than that in placenta, theca, and granulosa. Consistent with P450arom expression in these tissues, a cDNA encoding porcine P450arom hybridized to a 2.3-kb transcripts in Northern analyses of porcine blastocysts, placentas, and fetal and newborn adrenal glands and testes, as well as in theca and granulosa tissues from preovulatory follicles. No differences in transcript size were detectable among tissues. The identity of P450arom transcripts was confirmed by sequence analysis of partial cDNA clones amplified from porcine fetal adrenal glands, testes, and placentas according to the RACE procedure. The sequences of the adrenal and testis clones were identical but differed from the placental sequence, which represented the first 85 amino acids of porcine P450arom. Specifically, the adrenal and testis clones expressed transcripts that resembled the ovarian isoform of porcine P450arom, rather than the porcine placental isoform, predicting a two-amino acid deletion and 12 predicted amino acid substitutions. P450arom activity was examined to further define expression in these tissues. Activity in adrenal, testis, and placental homogenates was inhibited by 4-hydroxyandrostenedione (4OH-A4) whereas inhibition by etomidate was demonstrated in the adrenal and testis homogenates but not in the placental homogenates. The sensitivity of activity in the newborn porcine adrenal glands and testes to inhibition by etomidate was similar to that of ovarian P450arom activity. The level of P450arom activity was highest in the placenta and lowest in the adrenal gland, and no effect of fetal sex was noted in either tissue. Immunocytochemical studies localized the expression of P450arom in the adrenal gland of newborns to cells at the corticomedullary junction and, with greater intensity, to cells around the developing medullary lobules. The same cells expressed cytochrome P450 17 alpha-hydroxylase, the expression of which also extended throughout the zona fasciculata. The interstitial cells were the site of P450arom expression in the testis, but no expression could be detected in any cells with the spermatic tubules. These data demonstrate that fetal and newborn porcine adrenal glands and testes express an active P450arom that resembles the isoform expressed in the ovary. The localization of adrenal expression suggests a possible role in medullary maturation and function in the fetal and newborn pig.

Adrenal Glands↗
PubMed1996

Estrogen biosynthesis proximal to a breast tumor is stimulated by PGE2 via cyclic AMP, leading to activation of promoter II of the CYP19 (aromatase) gene.

In the present report, we show that prostaglandin (PG) E2 is the most potent factor which stimulates aromatase expression via cyclic AMP and promoter II. PGE2 acts via EP1 and EP2 receptor subtypes to stimulate both the PKC and PKA pathways. The combined stimulation of both of these pathways results in maximal expression of promoter II-specific CYP19 transcripts. Since PGE2 is a major secretory product both of breast tumor epithelial cells and fibroblasts, as well as of macrophages infiltrating the tumor site, then this could be the mechanism whereby estrogen biosynthesis is stimulated in breast sites adjacent to a tumor, leading in turn to increased growth and development of the tumor itself.

Adipose Tissue↗
PubMed1996

Aromatase expression in endometriosis.

The conversion of C19 steroids to estrogens occurs in a number of tissues, such as the ovary and placenta, and is catalyzed by aromatase P450 (P450arom; the product of the CYP19 gene). P450arom expression has also been detected in a number of uterine tumors, such as leiomyomas and endometrial cancer. On the other hand, P450arom expression was undetectable in normal endometrial and myometrial tissues. The present study was conducted to determine the presence or absence of aromatase expression in peritoneal endometriotic implants and in the eutopic endometrium of women with endometriosis. Endometriotic implants in pelvic peritoneum (n = 17; e.g. posterior culdesac, bladder, and anterior culdesac) and eutopic endometrial curettings (n = 11) of 14 patients with histologically documented pelvic endometriosis were obtained at the time of laparoscopy or laparotomy. Pelvic peritoneal biopsies distal to endometriotic implants as well as normal endometrial tissues (n = 7) from disease-free women were used as negative controls. We used competitive RT-PCR technology employing an internal standard to amplify P450arom transcripts in total ribonucleic acid (RNA) isolated from these tissues. P450arom transcripts were detected in all endometriotic implants and in all eutopic endometrial tissues from patients with endometriosis. P450arom messenger RNA species were not detectable in endometrial tissues from disease-free women or in endometriosis-free peritoneal tissues. The highest levels of transcripts were detected in an endometriotic implant that involved the full thickness of the anterior abdominal wall. The P450arom transcript level within the core of this endometriotic mass was 4-fold higher than that in the surrounding adipose tissue. It has been shown recently that aromatase expression in various human tissues is regulated by the use of tissue-specific promoters via alternative splicing. To analyze promoter usage, we amplified by RT-PCR the most likely promoter-specific untranslated 5'-termini of P450arom transcripts in 2 endometriotic implants. It appears that these endometriotic implants use both the adipose-type promoter I.4 and gonadal-type promoter II for aromatase expression. The use of promoter I.4 for aromatase expression in adipose tissue has been recently observed to be regulated by members of the interleukin-6 (IL-6) cytokine family. Based on these findings, we examined by RT-PCR, IL-6 and IL-11 messenger RNA expression in 5 endometriotic tissues and 1 eutopic endometrial sample from a patient with endometriosis. We detected IL-6 and IL-11 transcripts in all endometriotic tissues and in the eutopic endometrial tissue sample studied. Our findings indicate that both eutopic endometrial tissues and endometriotic implants from patients with endometriosis are biochemically different from normal endometrial tissues of disease-free women. The presence of aromatase expression in eutopic endometrial tissues from patients with endometriosis may be related to the capability of implantation of these tissues on peritoneal surfaces. Furthermore, the possibility of estrogen production in these implants may serve to promote their growth. Increased IL-6 and IL-11 expression in these tissues suggests that P450arom expression in endometriosis may be regulated in part by these cytokines.

Adult↗
PubMed1996

Use of alternative promoters to express the aromatase cytochrome P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients.

Estrogen biosynthesis in adipose tissue has assumed great significance in terms of a number of estrogen-related diseases. Recent evidence suggests that estrogen synthesized locally in the breast is of singular significance in the development of breast cancer in elderly women. The biosynthesis of estrogen from C19 steroids is catalyzed by a specific form of cytochrome P450, namely aromatase cytochrome P450 (P450arom; the product of the CYP19 gene). The human CYP19 gene comprises nine coding exons, II-X, and its transcripts are expressed in the ovary, placenta, testes, adipose tissue, and brain. Tissue-specific expression of the CYP19 gene is determined, at least in part, by the use of tissue-specific promoters, which give rise to transcripts with unique 5'-noncoding termini. Transcripts in adipose tissue contain 5'-termini derived from specific untranslated exons, corresponding to expression derived from the proximal promoter II and its splice variant I.3, as well as a distal promoter, I.4. The object of the present study was to determine the distribution of these various exon-specific transcripts in breast adipose tissues from cancer-free women undergoing reduction mammoplasty and from patients with breast cancer, because this would provide important clues as to the nature of the factors regulating aromatase expression in these sites. To achieve this, we employed competitive RT-PCR, utilizing an internal standard for each exon-specific transcript of the CYP19 gene, as well as for the coding region, to evaluate total CYP19 gene transcripts. In cancer patients (n = 18), total CYP19 gene transcript levels were significantly higher in adipose tissue proximal to a tumor in comparison with adipose tissue distal to a tumor, in agreement with previous findings. Moreover, total transcript levels were higher in breast adipose tissue of cancer patients in comparison with those of cancer-free individuals (n = 9), even when the adipose tissue from the cancer patient was taken from a quadrant with no detectable tumor. We observed that exon I.4-specific transcripts were predominant in breast adipose obtained from cancer-free women. In this tissue, promoter-II-specific and exon I.3-specific transcripts were present in low copy number. On the other hand, in breast cancer patients, CYP19 gene transcripts from breast adipose tissue had primarily promoter-II-specific and exon I.3-specific sequence, whereas comparatively few transcripts had exon I.4-specific sequence at the 5'-terminus. We conclude that CYP19 gene transcription in breast adipose tissue of cancer-free individuals uses preferably promoter I.4, implicating a role of glucocorticoids and members of the IL-6 cytokine family in the regulation of this expression. On the other hand, the increased expression in breast adipose tissue bearing a carcinoma results from expression from promoters II and I.3, which are regulated by unknown factors acting via increased cAMP formation, which are presumably secreted by the tumor or associated cells.

Adipose Tissue↗
PubMed1996

Distribution of aromatase P450 transcripts and adipose fibroblasts in the human breast.

The fibroblast component of adipose tissue is the primary extraglandular site of aromatase P450 (P450arom) expression, which is responsible for the conversion of C19 steroids to estrogens. Previously, we have shown positive correlations between the level of P450arom transcripts and the ratio of fibroblasts to mature adipocytes in adipose tissue samples proximal to breast tumors. The present study was conducted to determine the distribution of P450arom messenger ribonucleic acid (RNA) levels and fibroblast to adipocyte ratios in disease-free breasts of premenopausal women. Adipose tissue samples were collected from 3 regions (outer, upper, and inner) of both breasts of 13 women undergoing reduction mammoplasty. The histological composition of adipose tissue (n = 12) was determined by morphometry using a computerized image analysis program. We used a competitive RT-PCR method employing rat P450arom complementary RNA as an internal standard to quantify adipose P450arom transcripts in tissue total RNA samples (n = 11). Overall, 67% of the highest fibroblast to adipocyte ratios and 64% of the highest P450arom transcript levels were detected in an outer breast region, whereas in only 1 patient were the highest values detected in an inner region. Parametric ANOVA showed significant differences between the fibroblast content of the regions [p(F) = 0.037]. This distribution pattern directly correlates with the most common or the least common sites of carcinoma in the breast, the outer and inner regions, respectively. Moreover, a direct relationship was demonstrated between adipose fibroblasts and P450arom transcripts within the breast, in that regions with the highest fibroblast to adipocyte ratios contained the highest P450arom transcript levels (by ANOVA of contrast variables, P = 0.0009). These results suggest that, similar to our previous findings in the breast bearing a tumor, adipose tissue aromatase expression in the disease-free breast is determined by the local ratio of fibroblasts to adipocytes.

Adipose Tissue↗
PubMed1996

Cellular characterization of adipose tissue from various body sites of women.

Adipose tissue is the primary site of estrogen biosynthesis in postmenopausal women. The two main histologic components of adipose tissue are mature adipocytes and fibroblasts. Aromatase P450 expressed in the fibroblast component of adipose tissue is responsible for catalyzing conversion of C19 steroids to estrogens. We previously have demonstrated that in women, aromatase expression in adipose tissue of various body sites increases with age and that aromatase expression in the hip is markedly higher than in the abdomen. To determine whether this age- and regional-dependent variation in aromatase expression is caused by an alteration in the ratio of fibroblasts to mature adipocytes, we collected sc adipose tissue samples from 19 women (age range: 21-93 yr) at the time of autopsy. Using a computerized image analysis system, we determined by morphometry the proportions of adipocytes, fibroblasts, and vascular endothelial cells within histologic sections of adipose tissue from midabdomen, both breasts, and both hips. The percentage of each cell component at each body site was expressed as the mean of triplicate replicates. Statistical analysis of our results did not indicate any correlation between advancing age and fibroblast to adipocyte ratios in the breast, abdomen, or hip. Fibroblast to adipocyte ratios were found to be significantly higher in the breast and abdomen compared with the hip ( P < 0.05). No statistical differences were found between the breast and abdomen. These findings suggest that the increase in aromatase expression with advancing age and the higher aromatase expression in the hip compared with the abdomen in women may be caused by alterations in specific signal transduction mechanisms rather than a simple increase in local adipose fibroblast numbers.

Abdomen↗
PubMed1996

Tumor necrosis factor-alpha stimulates aromatase gene expression in human adipose stromal cells through use of an activating protein-1 binding site upstream of promoter 1.4.

Expression of aromatase P450 (P450arom; the product of the CYP19 gene) in human adipose stromal cells in primary culture is markedly stimulated by serum in the presence of dexamethasone (DEX). Under these conditions, the majority of P450arom transcripts contain untranslated exon 1.4 at their 5'-ends. Previously, we observed that the region of the CYP19 gene upstream of exon 1.4 contains a TATA-less promoter, a glucocorticoid response element, and an interferon-gamma-activating sequence. These act to mediate the action of interleukin-6 and related cytokines to stimulate aromatase expression in the presence of DEX. In the present study, we found that tumor necrosis factor-alpha (TNF alpha) also acts synergistically with DEX to stimulate aromatase expression in adipose stromal cells in serum-free medium. We observed that the action of TNF alpha can be mimicked by ceramide. Maximal aromatase activity was obtained when cells were incubated with 5 ng/ml TNF alpha or 100 nM ceramide in the presence of 250 nM DEX. Levels of c-fos and c-jun proteins also were increased by TNF alpha or ceramide in the presence of DEX. Upstream of the interferon-gamma-activating sequence site there is an imperfect activating protein-1 (AP-1) binding site (2-bp mismatch). Gel retardation analysis using nucleotide probes containing the putative AP-1-binding sequence and nuclear extracts of human adipose stromal cells cultured in the presence of TNF alpha or ceramide plus DEX revealed that adipose stromal cells nuclear proteins bind to this site and that binding was competed by a 100-fold excess of a consensus AP-1 sequence. In addition, binding activity was competed by both anti-c-fos and anti-c-jun sera. Mutation or deletion of the putative AP-1 element resulted in the loss of TNF alpha- plus DEX-induced activity of reporter constructs comprised of 515 bp of the exon 1.4 flanking sequence linked to the luciferase gene. These results suggest that TNF alpha, probably acting through ceramide formation, stimulates the binding of both c-fos and c-jun to the AP-1 element upstream of exon 1.4. These act cooperatively with the ligand-activated glucocorticoid receptor to induce aromatase expression in adipose stromal cells in primary culture. We conclude that this TNF alpha signal transduction pathway may play an important role in the regulation of estrogen biosynthesis in adipose tissue.

Adipose Tissue↗
PubMed1995

Aromatase P450 gene expression in human adipose tissue. Role of a Jak/STAT pathway in regulation of the adipose-specific promoter.

In the present report we describe a heretofore unrecognized role for a Jak/STAT signaling pathway, namely the stimulation of expression of the aromatase P450 (CYP19) gene, and hence of estrogen biosynthesis, in human adipose tissue. Expression of this gene in adipose tissue as well as in adipose stromal cells maintained in the presence of serum and glucocorticoids is regulated by a distal TATA-less promoter, I.4, which contains a glucocorticoid response element, an Sp1 binding site, and an interferon-gamma activation site (GAS) element. The stimulatory action of serum (in the presence of dexamethasone) can be replaced by interleukin (IL)-11, leukemia inhibitory factor, and oncostatin-M, as well as by IL-6, providing the IL-6 soluble receptor is also present. Stimulation of the cells by these factors led to rapid phosphorylation of Jak1, but not Jak2 or Jak3, on tyrosine residues. STAT3 but not STAT1 was also phosphorylated and bound to the GAS element in the I.4 promoter region. When regions of this promoter were fused upstream of the chloramphenicol acetyltransferase reporter gene and transfected into the cells, mutagenesis or deletion of the GAS element led to complete loss of reporter gene expression. Since adipose tissue is the major site of estrogen biosynthesis in men and in postmenopausal women, this pathway involving a Jak/STAT signaling mechanism acting together with glucocorticoids and Sp1 appears to be the principal means whereby estrogen biosynthesis is regulated in the elderly.

Adipose Tissue↗
PubMed1995

Ad4BP/SF-1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450 (CYP19) gene in the ovary.

Aromatase P450, which is responsible for the metabolism of C19 steroids to estrogens, is expressed in the pre-ovulatory follicles and corpora lutea of ovulatory women by means of a promoter proximal to the start of translation (PII). To understand how this transcription is controlled by cAMP, we constructed chimeric constructs containing deletion mutations of the proximal promoter 5'-flanking DNA fused to the rabbit beta-globin reporter gene. Assay of reporter gene transcription in transfected bovine granulosa and luteal cells revealed that cAMP-stimulated transcription was lost upon deletion from -278 to -100 base pairs, indicating the presence of a functional cAMP-responsive element in this region; however, no classical cAMP-responsive element was found. Mutation of an AGGTCA motif located at -130 base pairs revealed that this element is crucial for cAMP-stimulated reporter gene transcription. When a single copy of this element was placed upstream of a heterologous promoter, it could act as a weak cAMP-response element. Supershift electrophoretic mobility shift assay and UV cross-linking established that Ad4BP/SF-1 binds to this hexameric element. Ad4BP/SF-1 mRNA and protein levels and DNA binding activity are increased in forskolin-treated luteal cells. We conclude that cAMP-stimulated transcription of human aromatase P450 in the ovary is due, at least in part, to increased levels and DNA binding activity of Ad4BP/SF-1.

Animals↗
PubMed1995

Expression of a recombinant derivative of human aromatase P450 in insect cells utilizing the baculovirus vector system.

Aromatase P450 (P450arom) is the enzyme responsible for estrogen biosynthesis. Studies of the relationship of the function of this enzyme to its structure have been hampered by lack of a suitable preparation. In the present report we describe the expression of a recombinant derivative of P450arom in insect cells by means of the baculovirus vector system. This protein, which lacks the first 41 amino acids from the N-terminus, and hence the membrane-spanning region, has spectral properties and activity similar to that of the wildtype protein. Moreover, the presence of a hexameric histidine tag at the C-terminus permits its facile purification by means of nickel-agarose affinity chromatography. This system permits the synthesis of quantities of a biologically active derivative of P450arom suitable for studies designed to explore the relationship of function to structure.

Androstenedione↗
PubMed1995

A three-dimensional model of aromatase cytochrome P450.

P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane-bound proteins, they have resisted structure-function analysis by means of X-ray crystallographic methods. Among P450-catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three-dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox-partner binding, which require examination of the entire molecule (excepting the putative membrane-spanning region). This model of P450arom was built based on a "core structure" identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM-P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane-associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redox-partner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a "threonine switch" determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate.

Amino Acid Sequence↗