Eur J Biochem 269, 1393–1405 (2002) Ó FEBS 2002 Study of substrate specificity of human aromatase by site directed mutagenesis ´ P Auvray1,*,†, C Nativelle1,†, R Bureau2, P Dallemagne2, G.-E Seralini1 and P Sourdaine1 IBBA, Laboratoire de Biochimie et Biologie Mole´culaire, Universite´ de Caen, Esplanade de la Paix, Caen, France; CERMN, Laboratoire de Pharmacochimie, Caen cedex, France Human aromatase is responsible for estrogen biosynthesis and is implicated, in particular, in reproduction and estrogen-dependent tumor proliferation The molecular structure model is largely derived from the X-ray structure of bacterial cytochromes sharing only 15–20% identities with hP-450arom In the present study, site directed mutagenesis experiments were performed to examine the role of K119, C124, I125, K130, E302, F320, D309, H475, D476, S470, I471 and I474 of aromatase in catalysis and for substrate binding The catalytic properties of mutants, transfected in 293 cells, were evaluated using androstenedione, testosterone or nor-testosterone as substrates In addition, inhibition profiles for these mutants with indane or indolizinone derivatives were obtained Our results, together with computer modeling, show that catalytic properties of mutants vary in accordance with the substrate used, suggesting possible differences in substrates positioning within the active site In this respect, importance of residues H475, D476 and K130 was discussed These results allow us to hypothesize that E302 could be involved in the aromatization mechanism with nor-androgens, whereas D309 remains involved in androgen aromatization This study highlights the flexibility of the substrate–enzyme complex conformation, and thus sheds new light on residues that may be responsible for substrate specificity between species or aromatase isoforms Estrogens are known to be implicated in reproduction and estrogen-dependent tumor proliferation [1] Moreover, an abnormal expression of aromatase, the enzyme involved in the conversion of androgens to estrogens, has been detected in breast tumors and in surrounding adipose stromal cells [2,3] The aromatase enzyme comprises a specific cytochrome P-450 aromatase and the ubiquitous cytochrome P-450 NADPH reductase A common treatment for estrogen-dependent cancers is the use of antiestrogens and/or aromatase inhibitors [4] The usual way to develop new aromatase inhibitors is to screen in vitro chemical compounds [5–10] from the knowledge of the substrate structure, or by comparisons with other aromatases [11–13] The design of more specific and efficient aromatase inhibitors could be improved by a better knowledge of the enzyme’s active site A precise modeling of this part of the molecule is therefore necessary Despite success in obtaining aromatase purified to homogeneity [14], crystallization of this microsomal membrane-anchored protein has not been reported Knowledge of the aromatase structure is largely derived from comparisons with soluble bacterial cytochromes Pseudomons putida camphor P-450 (P-450cam), Bacillus megaterium P-450 (P-450BM-3), Pseudomonas putida a-terpineol P-450 (P-450terp) and Saccharopolyspora erythreae erythromycin F P-450 (P-450eryF), these proteins being well characterized and crystallized [15–19] However, human aromatase shares only 15–20% homology with these bacterial cytochromes A theoretical molecular model of P-450arom has been proposed [20], but the model revealed a poor energy profile in the regions between residues 150–250 [21]; the problems seemed to be attributed to the length of helices F and G, and a model based on cytochrome P-450cam is better defined in this region [21] It is very difficult to produce a more reliable model, irrespective of the bacterial cytochrome P-450 used for alignment In fact, these later cytochromes P-450, with resolved three-dimensional structures, have very weak sequence homologies with P-450arom Moreover, FASTA3 AND BLAST analyses of the protein databank sequences not provide more reliable models of amino-acid sequences Studies using site-directed mutagenesis provides a way of validating partial or complete models Such structure–function studies make it possible to identify important regions directly or indirectly implicated in the aromatization mechanism These domains are the substrate access channel (constituted by the b 1-1 sheet), the FG loop and the aromatase specific region [20] Graham-Lorence et al [20] suggested that the B¢C loop has an important function in substrate orientation, and that ´ Correspondence to P Sourdaine, IBBA, Universite de Caen, Esplanade de la Paix, 14032 Caen cedex, France Fax: + 33 31 56 53 20, Tel.: + 33 31 56 53 70, E-mail: bioch.bio.mol@ibba.unicaen.fr Abbreviations: hP-450arom, human P-450 aromatase; eP-450arom, equine P-450 aromatase; P-450cam, P450 camphor from Pseudomons putida; P-450BM-3, P450 from Bacillus megaterium; P-450terp, P-450 a-terpineol from pseudomonas putida; P-450eryF, P-450 erythromycin F from Saccharopolyspora erythreae; 4-OHA, 4-hydroxyandrostenedione Enzyme: cytochrome P450 aromatase (EC 1.14.14.1) *Present address: Oncodesign S.A., Parc Technologique de la Toison d’Or, 21000 Dijon, France  Note: these authors contributed equally to this work (Received 17 December 2001, accepted 11 January 2002) Keywords: aromatase; site-directed mutagenesis; molecular modeling; androgens; inhibitors 1394 P Auvray et al (Eur J Biochem 269) a part of the b4 sheet (K473–D476) is implicated in the substrate pocket, particularly in the extrahydrophobic pocket [21] Previous site-directed mutagenesis studies suggest a role for specific residues such as E302, D309, T310 [20–24] and K473, H475 [20,25] Finally, the regions involved in redox-partner have been defined as being in B, C, J, J¢, K and L helices Recently, the report of three isoforms of porcine aromatase, encoded by distinct genes [26] and the study of their catalytic differences [27,28] also highlight the need to understand which residues of the enzyme are involved in the substrate specificity In addition to human and porcine aromatases, equine aromatase is also well characterized biochemicaly [29–34] For example, nor-testosterone is more rapidly aromatized by the equine aromatase, despite a weaker affinity, than the human enzyme [34]; some inhibition differences have also been described [11,13,31] Taking into account these results and the three-dimensional model of both human and equine P-450arom we have suggested that H475 and D476 have a role in the interaction of the indane derivative MR 20814 within the active site [11] Therefore, the aim of our present study was to explore the role of H475 and D476 and of other human aromatase residues which might be involved in the orientation and binding of substrates Human residues were mutated to the corresponding aligned equine residues (Fig 1), apart from D309 and E302, which have been extensively studied in the literature H475, which is Asn in horse and other species, and D476 which is absolutely conserved, were more extensively studied because of their location within an extrahydrophobic pocket, or a hydrophobic surface [21] This could determine differences in inhibition between human and equine P-450arom by MR 20814 [11] EXPERIMENTAL PROCEDURES Chemicals All chemical products were obtained from Sigma (St Quentin Fallavier, France) (polyethylenimine, 50 kDa, was prepared in ddH2O at 10 mM, pH 7.0) or GibcoBRL (Cergy Pontoise, France) [1b,2b-3H]Androstenedione was Fig CLUSTALW 1.81 multiple sequence alignment of human and equine aromatases Human and equine sequences were, respectively, from Corbin et al [35] and Tomilin et al [33] Ó FEBS 2002 from Dupont NEN (Les Ulis, France), testosterone and 19nor-testosterone from Sigma (St Quentin Fallavier, France), solvents from Carlo Erba (Val de Reuil, France) and sds (Peypin, France), 293 cells (ECACC number: 85120602) with stable expression of cytochrome P450 reductase ´ (Kindly provided by V Luu-The, CHUL, Quebec), QuickChangeTM Site-Directed Mutagenesis kit from Stratagene (Montigny le Bretonneux, France), alkaline phosphatase substrate kit from Bio-Rad (Ivry sur Seine, France), culture media from BioWhittaker (Gagny, France), Thermo Sequenase Kit from Amersham (Les Ulis, France), pCMV plasmid from Invitrogen (NV Leek, the Netherlands) and Qiagen Plasmid Maxi Kit from Qiagen (Courtaboeuf, France) Human aromatase cDNA was kindly provided by E R Simpson (Monash University, Melbourne, Australia) The oligonucleotide primers were from Pharmacia (Orsay, France) or EUROBIO (Les Ulis, France) Indane and indolizinone derivatives were produced by the CERMN (Caen, France, Fig 2) PCMV-human aromatase cDNA construction The plasmid used in this study has been previously described [13] Briefly, human aromatase cDNA (2920 bp) [35] was cloned into pUC18 (2.7 kb) with two fragments of kgt10 HindIII–EcoRI at the 5¢ end (240 bp), and EcoRI–BglII at the 3¢ end (900 bp) This construction was partially digested with EcoRI and the EcoRI–EcoRI fragment (2920 bp) was cloned into pCMV (EcoRI site at position 753) The construction orientation was checked by sequencing and by specific PCR amplification with the primers sense A (555–575, 5¢-CCATTGACGTCAATGGGAGTT-3¢) and antisense B (1920–1899, 5¢-TAAGGCTTTGCGCATGAC CAAG)3¢), which are specific to pCMV and the cDNA, respectively (the amplicon length was 1368 bp) The pCMV-cDNA was purified from JM109 bacterial strain amplification by Qiagen Plasmid Maxi Kit The length, concentration and purity of the plasmid-cDNA construction were checked by 1% agarose electrophoresis and ethidium bromide staining Fig Structure of inhibitors 4-OHA is from Brodie et al [64], MR 20814, MR 20492 and MR 20494 from Auvray et al [11,13] Ó FEBS 2002 Structure–function relationships of human aromatase (Eur J Biochem 269) 1395 Aromatase cytochrome P-450 molecular modeling Initial alignments of cytochrome P-450 BM3 and aromatase cytochrome P-450 were taken from the alignments of Graham-Laurence et al [20] Main chain coordinates for the core regions were taken directly from the cytochrome P-450BM3 structure Using coordinates for the loops obtained from the loop data base search The replace residue command was used to replace a residue In this case, the replacement residue is first aligned to the backbone of the original residue After the backbone has been aligned, the dihedral angles in common with the residue being replaced are also aligned New charges and potential functions types were taken from the residue library Refinement of the structures involved energy minimization using AMBER [36] and ESFF FORCE FIELD (Program DISCOVER version 95, http://www.accelrys.com/support/life/discover/ forcefield/esff.html) [37,38] Difficulties modelling the heme led to the choice of ESFF force field for this region As far as possible, the atomic parameters were directly determined from experimental or calculated rather than fit For the valence energy in this force field, as with AMBER, only diagonal terms were included The partial charges were determined by minimizing the electrostatic energy with respect to the charges, with the constraint that the sum of the charges is equal to the net charge on the molecule In this case, electronegativity and hardness were determined ESFF was based on electronegativities and hardnesses calculated using the density functional theory For the van der Waals interactions, ESFF used the 6-9 potential The van der Waals parameters were derived using rules consistent with the charges The minimization algorithms used were Steep˚ est Descents until a gradient of 10 kcalỈmol)1ỈA)1, then ˚ conjugate gradients until a gradient of kcalỈmol)1ỈA)1 The final structure was analyzed by PROCHECK [39] and PROSA [40] No specific routine were used for docking of substrate and inhibitors Docking of the androstenedione into the active site was carried out considering the orientation of C(1), C(2) and C(19) above the heme [41] and the position of the ligand towards D309 and T310 The threedimensional structure of androstedione was obtained from crystallographic data [42] This docking was followed by a minimization to refine the complex This method produced difficulties in maintaining the overall three-dimensional structure of the steroid Indeed, during the minimization, steric interactions between V370 and the A ring of the androstenedione led to a modification of the conformation of this ring We optimized the position of the ligand and the orientation of the hydrophobic group of V370 (modification of dihedral angles) to decrease this interaction V370 is highly conserved suggesting an important role of this residue in the active site For the inhibitors, we have considered an orientation of the pyridine group towards the heme and of the amine group towards the extrahydrophobic surface A discussion on this proposal of docking and on the conformation of the inhibitor was carried out as described previously [11] Noncovalent interactions between different residues or between residues and substrates were determined using the ISOSTAR software [43] Theoretical noncovalent interactions from the ISOSTAR software were calculated from the sum of the following terms: the electrostatic energy (attractive and repulsive Coulombic interaction); the exchange–repulsion term (sum of an energy lowering due to exchange of electrons of parallel spin between the molecules and the repulsive term arising from the Pauli exclusion principle); the polarization energy (energy gain caused by the change of intramolecular wave function of one molecule due to the presence of the undistorted charge distribution of the second molecule); the charge-transfer energy (attractive energy from actual charge transfer between molecules); the dispersion energy (calculated at the second order double excitation level) The definition of the lipophilicity potential (MOLCAD SURFACE; program SYBYL 6.0; Tripos Association: St Louis, MO, USA) is calculated on the basis of the atomic partial lipophilicity values [44] and a distance-dependent function [45] Site-directed mutagenesis This step was performed with the QuickChangeTM SiteDirected Mutagenesis method from Stratagene Briefly, this was based on a PCR with two complementary oligonucleotide primers containing the mutation The PCR was performed with the Pfu DNA polymerase during 16 cycles (30 s at 95 °C, 30 s at 55 °C and 13 at 68 °C) The PCR products were then digested with DpnI which only digests the parental methylated cDNA Nicked vector DNA with the desired mutations was then transformed into Escherichia coli XL1-Blue supercompetent cells Transformed bacteria were analyzed directly on colonies by PCR with primers 5H1 (1361–1379, 5¢-GTCGTGTCATGCTGGACAC-3¢) and 3H-54 (2384–2367, 5¢-GAGGATGACACTATTGGC-3¢) after 30 cycles (1 at 95 °C, at 52 °C, at 72 °C; cycle: 10 at 72 °C) The expected amplicon length was 1026 bp Mutations were then checked by sequencing 10 lL bacterial DNA miniprep with the Thermo Sequenase, as previously described [13] Plasmid DNA was extracted as follows: the bacterial pellet was lysed with 8% sucrose, 0.5% Triton X-100, 0.05 M EDTA, 0.01 M Tris/HCl pH 8.0 and 10 mgỈmL)1 lysosyme/0.01 M Tris/ HCl pH 8.0, boiled and the DNA was then precipitated by 3M NaOAc pH 7.0 and isopropanol After sequencing, pCMV-cDNA was purified from XL1-Blue supercompetent bacterial strain amplification by means of the Qiagen Plasmid Maxi Kit, as previously described Culture and transfection of 293 cells Cells were grown in red phenol-free EMEM medium and supplemented with mM glutamine, 10% new-born calf serum (supreme serum), 1% nonessential amino acids at 37 °C in an atmosphere of 5% CO2 and 95% air Cells (50 000) were grown to 50% confluence on 24-well cell culture plates 18 h before transfection, washed with serum-free cell culture medium, supplemented with 500 lL serum-free medium and transiently transfected with lg pCMV-human aromatase cDNA, using a modification of the method of Boussif et al [46] Briefly, lg pCMVcDNA (6 nmol of phosphate) and 54 nmol of polyethylenimine were separately diluted with 50 lL 150 mM NaCl, incubated for 10 at room temperature in a laminar fume hood, mixed together, incubated for another 10 at room temperature, and then added to each well Cells were incubated for 3–4 h at 37 °C and then supplemented with Ó FEBS 2002 1396 P Auvray et al (Eur J Biochem 269) 500 lL medium containing 10% supreme serum After a further 18-h incubation, cells were washed with serum-free medium and the aromatase activity was measured in whole cells Evaluation in the whole cell rather than the microsomal aromatase activity was used because it may allow better approximation in the in vivo situation [21] ELISA quantification of the aromatase expressed was used as an indicator of the transfection efficiency Whole cell aromatase activity and inhibition Aromatase activity was assessed in whole cells using the method described by Zhou et al [47] by measuring the H2O released from [1b,2b-3H]-androstenedione Cells were washed with serum-free culture medium Dessicated radioactive substrate (200 nM and 50–800 nM for IC50 and kinetic experiments, respectively) supplemented with lM progesterone (used to block 5a-reductase activity) and 0–10 lM inhibitor (for IC50 experiments) were mixed with serum-free culture medium and added to each well Cells were incubated at 37 °C under 5% CO2 for 45 After incubating cells for on ice, the culture medium (1 mL) was sampled and extracted by CHCl3 (1 mL) Steroids were then removed by incubation with mL charcoal dextran suspension (7%/1.5%), and the radioactivity of the aqueous phase was measured as previously described The results were the mean of at least triplicate experiments ± SD and were expressed as pmolỈmin)1Ỉmg aromatase)1 Results from control incubations, produced by transfecting under the same conditions the pCMV plasmid alone instead of the pCMV-cDNA plasmid, were used to determine the limit of detection Km.app and Vm.app determinations were carried out using linear regression analysis of both Lineweaver–Burk and Hanes–Wolf plots Steroid radioimmunoassays The 17b-estradiol was assayed in 293 cells supernatant after incubation times of 45 (nor-testosterone) or 90 (testosterone) with 50–1600 nM substrate in the same conditions as those described above, and after extraction with 10 volumes of diethyl ether as previously described [48] The supernatant was chosen after demonstrating that the total part of steroids was in this compartment (data not shown) Estradiol rabbit antibodies [(66033) 3H-estradiol ´ ` RIA kit, bioMerieux, Charbonnieres les Bains, France] were diluted twofold according to the manufacturer’s instructions The extraction efficiency was 80 ± 5% and the sensitivity of this radioimmunoassay was 10 pgỈmL)1 Results, calculated according to Garnier et al [49], were the mean of at least triplicate experiments ± SD and are expressed as pmolỈmin)1Ỉmg aromatase)1 Enzyme-linked immuno-sorbent assays Cells were scraped from culture wells (pools of three culture wells), resuspended in 500 lL of water and sonicated on ice twice at 40 Hz for 20 s Aromatase in transfected cells was evaluated by a direct sandwich ELISA method adapted to our model: a 200-lL cell homogenate or 200 lL of NaCl/Pi containing 2–8 ng of purified equine aromatase (standard curve) were mixed with 800 lL of polyclonal antibody Fig Standard curve of ELISA (A) The standard curve was obtained by mixing 200 lL NaCl/Pi containing 0–8 ng of purified equine aromatase to 800 lL of polyclonal anti-(ep.450arom) Ig; : 10 000 The fixation of the primary antibody was then evaluated with anti-(rabbit IgG) Ig coupled to alkaline phosphatase and incubation with p-nitrophenylphosphate Absorbance was read at 405 nm on a Bio-tek EL 800 apparatus (B) Westernblot analysis for P450arom Westernblotting of P450arom in mock E293 cells and E293 cells transfected with lg pCMV-human aromatase cDNA The size and position of the expected 55 kDa aromatase protein is shown (1 : 10 000), raised against intact eP-450arom [29], incubated for h and then added (100 lL per well) to plates (Nunc, high protein adsorption quality) Plates were previously coated overnight at °C with 50 ng per well of purified equine aromatase, saturated h at 37 °C with 200 lL NaCl/Pi/Tween 20 (0.1%)/gelatin (0.5%) and washed with 150 lL NaCl/Pi/Tween 20 (0.1%) The fixation of the anti-(eP-450arom) Ig was then evaluated by incubating for h at 37 °C with 100 lL of anti-(rabbit IgG) Ig coupled to alkaline phosphatase (1 : 6000), washing and incubating for 1.5 h at 37 °C with 100 lL of the substrate p-nitrophenylphosphate as described by the manufacturer The absorbance was finally read on a Bio-Tek EL800 apparatus (Packard) at 405 nm Results were the mean of triplicate experiments ± SD and are expressed in ng aromatase per culture well Sensitivity of the assay was 0.2 ng per well of ELISA (Fig 3) corresponding to 1.6 ng per well The antiequine aromatase polyclonal antibodies were prepared in our laboratory [29] The total protein quantity was evaluated according to Bradford [50] Statistical study Data were compared using the Mann–Whitney test (ANOVA) RESULTS The catalytic properties of P450arom mutants are summarized in Tables and Results from an investigation of the interaction of different aromatase inhibitors with mutants, to test the accuracy of our computer model as well as to understand the inhibition characteristics of nonsteroidal inhibitors (MR 20814, MR 20492 and MR 20494) are shown in Fig IC50 are presented in Table and are analyzed in respect of catalytic properties of mutants with the substrates tested Western blot analysis [29] demonstrated that the polyclonal antibodies specifically detected human aromatase in microsomes Aromatase was also evident in E293 cells Ó FEBS 2002 Structure–function relationships of human aromatase (Eur J Biochem 269) 1397 Table Kinetic parameters of wild-type and mutant forms of P450 aromatase using androstenedione as a substrate The human residues were mutated in different domains of the protein, sometimes by their corresponding aligned equine residues A: Mutations of residues conserved in both species B: Non conservative changes C: Conservative changes Cells were transfected by human P450arom cDNA and the aromatase activity was evaluated by the tritiated water assay using [1b,2b-3H]-androstenedione as a substrate, as described in Materials and methods The aromatase quantity was evaluated by ELISA in order to correct the aromatase activity for transfection efficiency Results are the mean of at least three experiments in triplicate ND, activity not detectable NC, activity too low to calculate kinetic parameters (NC1, NC2 and NC3: activity below 1%, 5% and 15%, respectively, relative to wild-type with 200 nM of androstenedione corresponding to an activity of 1497 ± 300 pmolỈmin)1Ỉmg aromatase)1) Protein B C a P < 0.05 b Wild-type Km (%) Vm.app (pmolỈmin)1Ỉmg)1) Wild-type Vm (%) Wild-type E302A D309A D476A D476K D476L D476N D476E K119T K119Y K119V K119E C124Y K130N F320C H475N H475R H475A H475E I125M S470N I471M I474T A Km.app (nM) 164 ± NC1 NC1 NC1 NC1 NC2 186 ± 125 ± NC1 472 ± 179 ± 57 ± 151 ± 147 ± 111 ± NC2 NC2 68 ± NC2 ND 290 ± NC3 635 ± 38 100 ± 23 824 100 ± 27 71 50 113 ± 43 76 ± 30 605a 657 56 ± 19 87 ± 21 51a 84 7a 47 20a 287 109 34 91 89 67 3041 ± NC1 NC1 NC1 NC1 NC2 1727 ± 2671 ± NC1 3240 ± 4864 ± 4064 ± 1496 ± 2473 ± 2127 ± NC2 NC2 2388 ± NC2 ND 2443 ± NC3 4107 ± ± ± ± ± ± ± 30 51 28 12 16a 41 ± 10 104a 176 ± 63 251c 386 ± 152 1461 301b 623 302a 1465 782 106 159 133 49 81 69 ± ± ± ± ± ± 48 20 48 25 1518 78 ± 49 587 80 ± 19 1583 135 ± 52 P < 0.005 c P < 0.001 (ANOVA) transfected with the pCMV-human aromatase cDNA construct (Fig 3) All mutants could be detected in the 293 transfected cells using ELISA with an average content per culture well of 3.08 ± 1.58 ng (mean ± SEM, n ¼ 305), 2.33 ± 0.9 ng for the wild type (n ¼ 68) and with a range of 2.11 ± 0.6 ng for D476A (n ¼ 5) to 4.48 ± 2.63 ng for K130N (n ¼ 33) Aromatase activity was expressed per mg of P450-arom in order to take into account the turnover and the expression rate of the protein [51] H475 and D476 residues H475 or D476 were mutated (Table 1) to determine the importance of the residue nature at these positions With androstenedione as substrate, six mutations strongly decreased aromatase activity: H475N, H475R, H475E, D476A, D476K and D476L (activities below or 5% relative to wild-type with 200 nM of androstenedione, Table 1) In contrast, the Km.app value for H475A and the Vm.app value for D476N decreased and values for D476E were not different from those of wild-type enzyme D476N and D476E were also tested with testosterone and nortestosterone as substrates (Table 2) The decrease of the binding affinity of testosterone for mutant D476N was accompanied by a decrease in the Vm.app value although catalytic properties of D476N were unchanged with nortestosterone A lower aromatase activity with these substrates was observed for D476E (13% and 8% of wild-type for testosterone and nor-testosterone, respectively), contrasting with the results obtained with androstenedione The relative potency of the three inhibitors tested, according to their IC50 values (Table 3), was increased for inhibition of H475A, which had a lower Km.app value for androstenedione than that of the wild-type aromatase IC50 values showed D476N to be more sensitive to MR 20814 and MR 20492 than the wild-type D476E showed differences in aromatase activity according to the substrate used, and responded differently to MR 20814 and MR 20492 Domains of the active site and substrate specificity D309 was predicted to be directly involved in decarboxylation and aromatization mechanisms [20], and its mutation to Ala induced an activity loss whether androstenedione or testosterone was substrate However, D309A had activity with nor-testosterone (Table 2) with Km.app and Vm.app values similar to those of the wild-type enzyme Furthermore, E302A was inactive with all substrates tested Human residues implicated in the active site of the aromatase were mutated to corresponding aligned equine Ó FEBS 2002 1398 P Auvray et al (Eur J Biochem 269) Table Kinetic parameters of wild-type and mutant forms of P450 aromatase using testosterone or nor-testosterone as substrates Cells were transfected by human P450arom cDNA and the aromatase activity was evaluated by radioimmunoassay of 17b-estradiol, using testosterone or 19 nor-testosterone as substrates, as described in Materials and methods The aromatase quantity was evaluated by ELISA in order to correct the aromatase activity for transfection efficiency Results are the mean of at least three experiments in triplicate, except for D476N and M85V with testosterone (n ¼ 2) ND: activity not detectable; NC: activity too low to caculate kinetic parameters (NC1, NC2 and NC3: activity below 1%, 5% and 15%, respectively, relative to wild-type with 200 nM of testosterone corresponding to an activity of 1731 ± 180 pmolỈmin)1Ỉmg arom)1 or with 200 nM of nor-testosterone corresponding to an activity of 2287 ± 477 pmolỈmin)1Ỉmg arom)1) Testosterone Nor-testosterone Protein Km.app (nM) Vm.app (pmolỈmin)1Ỉmg)1) Km.app (nM) Vm.app (pmolỈmin)1Ỉmg)1) Wild-type K119E K130N E302A D309A F320C S470N D476N D476E 238 ± 256 ± 76 ± ND NC1 NC1 NC2 480 ± NC3 4593 ± 1508 ± 1610 ± ND NC1 NC1 NC2 2661 ± NC3 358 ± – 791 ± ND 301 ± 386 ± 338 ± 414 ± NC3 4531 ± – 835 ± ND 4662 ± 2165 ± 3494 ± 3850 ± NC3 a 19 29 21a 91 613 297a 88a 258 140a 57 137 44 41 887 179a 2173 698a 269a 512 P < 0.05 (ANOVA) Table IC50 values with steroidal and nonsteroidal aromatase inhibitors used with human aromatase mutants Aromatase activity was evaluated by measuring the amount of 3H2O released from 200 nM [1b,2b-3H]-androstenedione incubated in culture medium at 37 °C-5% CO2 atmosphere for 45 in presence of inhibitors Aromatase activities were expressed as percentage of a standard control which was incubated without inhibitor in the same conditions IC50 values in lM, were the mean ± SD of three (wild-type) or two (mutants) experiments in triplicate IC50 value with 4OHA, used as control, was 0.45 ± 0.35 lM with the wild-type protein IC50 (lM) Protein MR20814 MR20492 MR20494 Wild-type K119Y K119V K119E C124Y K130N F320C I474T H475A D476N D476E 10.83 ± 3.76 ± > 10 9.80 ± 4.40 ± 0.23 ± > 10 > 10 0.92 ± 2.02 ± 4.68 ± 3.93 ± 5.72 ± 6.21 ± 0.74 ± 0.33 ± 4.27 ± 5.35 ± > 10 0.45 ± 0.92 ± 5.60 ± 0.23 0.17 1.55 0.53 0.33 0.20 0.94 2.80 0.10 0.15 0.15 a 46 0.80 1.68a 0.42 1.97a 0.16a 0.12a 1.44a 0.12a 0.93 1.10 2.60 0.17a 0.25a 2.58 1.12 0.007a 0.12a 0.28a ± ± ± ± ± ± ± ± ± ± ± 0.02 0.03 0.5a 0.45 0.18 0.13 0.50a 0.57a 0.00a 0.07 0.07 P < 0.05 (ANOVA) residues The mutant K130N had similar catalytic properties for androstenedione when compared to the wild-type enzyme (Table 1) K119T also had greatly reduced aromatase activity as was also observed for C124Y (but to a lesser extent), which had a lower Vm.app value F320C increased the affinity for the substrate Studies of the nature of the residue at position K119 showed that K119Y greatly decreased the binding affinity for androstenedione, whereas K119E increased it and K119V increased the Vm.app value for androstenedione Interestingly, the binding affinity of K119E for testosterone was unchanged when compared to the one of wild-type P450arom but the Vm.app decreased Catalytic properties of the mutant K130N for testosterone and nor-testosterone were different from those observed for androstenedione When compared to the wild-type enzyme, this mutant was found to have a higher binding affinity, but a lower Vm.app value, for testosterone whilst its binding affinity and the Vm.app value were lower for nor-testosterone The mutant F320C, weakly active with testosterone, was found to have a lower Vm.app value for nor-testosterone than that of the wild-type P-450arom K130N and K119E produced a greater inhibition with MR 20814 or MR 20492, respectively, whereas F320C and K119V decreased the inhibition potency of MR 20494 (Table 3) Furthermore, Table indicates that I125 and I471 could be directly or indirectly implicated in the active site structure since their mutations produced weak or inactive proteins The Km.app values for I474T and S470N increased compared to the wild-type enzyme Although S470N had a lower binding affinity for androstenedione, it showed only 2% of wild-type aromatase activity for testosterone and a slightly lower Vm.app value for nor-testosterone The inhibition study with these mutants (Table 3) revealed that I474T, had decreasing affinity for androstenedione and was less inhibited by the three molecules tested DISCUSSION Based on the aromatization characteristics and inhibition of our human mutants, the role of each residue studied may be discussed taking into account previous knowledge of the biochemistry of equine aromatase [30–32] and the documented model of Graham-Lorence et al [20] together with results from our molecular model The Km.app of the recombinant wild-type aromatase for androstenedione (164 ± 38 nM) is in the range of Km values reported in the literature (9–150 nM) [14,21,24,52–54] In our study, the Km.app for testosterone is slightly higher than for androstenedione, as was observed by other groups Ó FEBS 2002 Structure–function relationships of human aromatase (Eur J Biochem 269) 1399 Fig Enzymatic mechanism of the human aromatase with androgens (A) and nor-androgens (B) The enzymatic mechanism was modified from Graham-Lorence [20] and from Ahmed [41,59] [14,55,56], whereas other studies have reported similar values for both substrates [14,35,50] Furthermore, we found a 1.5-fold increase in Km.app value for nor-testosterone when compared to the value obtained for testosterone These results are in accordance with those obtained by Kellis & Vickery [55] for nor-androstenedione and androstenedione Despite the fact that the antibodies used in this study are polyclonal and also recognize the human aromatase (Fig 3) [29], we cannot exclude that the antibodies bind the human and the equine enzymes with different affinities Therefore, the comparison of the absolute values of Vm obtained in this study with those reported in the literature is more difficult By using ELISA, we found aromatase in human placental microsomes at 210 pmolỈmg proteins)1, which is  2.5-fold higher than the results reported by Kadohama et al [51] Furthermore, the turnover rate observed in our study for the recombinant wild-type aromatase is of 0.2 min)1, which is 10-fold lower than reported by Chen et al [23] in CHO transfected cells but is in the lower range observed for human purified aromatase (0.6–35 min)1) [14,57,58] Only significant differences of Vm.app between the wild-type and the mutants will be discussed Enzymatic mechanism The mutation D309A has been already described and the probable role of D309 is to bring a proton at C(19) (decarboxylation) and attract a proton at C(2) (aromatization), helped by the basic residues H475 or K473 near C(3) [20] Moreover, Ahmed [41,59] proposed a mechanism for the aromatization by ferroxy radical attack on C(19) (Fig 4A) According to Graham-Lorence et al [20], E302, which was previously thought to interact with the substrate [60], is too far from C(2) Therefore, these authors suggested that D309 was a candidate to attract a proton at the C(2) position This hypothesis has been checked by mutating D309 to Ala or Asn Moreover, Zhou et al [52] did not demonstrate any 19-hydroxy and 19-oxo intermediates with D309A and D309N These results supported D309 acting as proton donor to T310 during the first ferroxy radical formation However, our results showed that D309A was only active with 19-nor-testosterone, suggesting that the aromatization mechanism of nor-androgens was different From the model, hydrophobic interactions were observed between the C(19) of androgens and a hydrophobic surface formed by the residues F134 and K130 (alkyl chain) The absence of C(19) for nor-androgens, reducing steric constraints, could allow the C(2) of nor-testosterone to come closer to E302 The aromatization of nor-androgens (Fig 4B) could then be different, with a first step corresponding to the ferroxy radical formation, the second step corresponding to the attack of C(1) by this radical to hydroxylate this position, the third step corresponding to the loss of H2O and the final step corresponding to the aromatization of cycle A, as previously described by Ahmed [59] The loss of activity of E302A with nor-testosterone as a substrate supported this hypothesis From this proposed mechanism, a single hydroxylation at C(1) would be sufficient to allow the aromatization of norandrogens with a 1-hydroxylated intermediate compound, as was already suggested by Ganguly et al [61] 1400 P Auvray et al (Eur J Biochem 269) Ó FEBS 2002 In their recent study, Kao et al [62] emphasized the importance of the interaction between E302 and androstenedione based on a decrease in the Vm value for the mutant E302D due to a modification of the active site size Similarly, our results show that the mutant E302A is not active with the three substrates tested These observations also emphasize the importance of this residue in the active site structure However, we propose a slightly different role for E302 in the structure–function relationships of aromatase than that put forward by Kao et al [62] On the other hand, the K473 position within the active site and our kinetic results obtained with H475 mutants (see below), makes the role of these residues in the enolization of 3-keto group unclear Our hypothesis is that the driving elements for the aromatization are the acidic properties of C2 hydrogens (in a position of the keto group) and the electrostatic attractions of D309 towards these hydrogens The substrate-binding pocket Residues toward the keto groups (a-face) of the steroid – D476 We previously suggested that position 476 could be important in the active site [11] We then mutated D476 in order to understand the role of this residue Results showed that position 476, and more particularly an acidic residue, appeared to be important for the aromatase activity Like D309, D476 may interact with the C(16) hydrogens (Fig 5) in a position of the keto group [C(2) hydrogens for D309] This electrostatic interaction, with an acidic group, seems to be an important part of the stabilization of the steroid inside the active site (theoretical noncovalent interactions of )67 kJỈmol)1 [43]), as suggested by the results of the mutations D476A, D476K and D476L leading to inactive enzyme vs D476E mutation that did not modify Km.app and Vm.app values with androstenedione However, with testosterone and nortestosterone, the D476E mutation produced an inactive compound In this last case E476, by its longer lateral chain than D476 and by a straight interaction with the hydroxyl group (calculated non covalent interaction of )77 kJỈmol)1 with ISOSTAR software), could destabilize the position of these ligands inside the active site When compared to the wild-type enzyme, D476N had lower Vm.app values for C19-androgens but similar Km.app and Vm.app values for nor-testosterone These observations suggest that this mutation produces a modification of the interaction between C(2) and D309 during aromatization of the C19-androgens D476 and D309 line the same face of the active site and the repulsive force (electrostatic interactions) between these two acidic residues appears to be important for efficient aromatization of C19-androgens This is also supported by results obtained with D476A, D476K and D476L Moreover, because E302 is located on the opposite face of the active site, aromatization of nor-testosterone was unchanged All these results suggest that D476 protrudes into the active site and may interact with the substrate For MR20814, we have previously shown [11] the existence of a coordination bond between the pyridine group and iron (Soret band) Because of this, we have suggested the following position of the ligand inside the active site (Figs and 7) corresponding to an orientation of the amino group towards the extrahydrophobic surface composed of residues I474 and L477 [21], an interaction between F134 and the Fig Relative position of residues toward androstenedione in the active site D476 and D309 may stabilize androstenedione by interacting with the C(16) and C(2) hydrogens, respectively K130 by its long aliphatic lateral chain will reinforce hydrophobic interactions over the b face of the steroid The quadropole/quadropole interaction of the amino group of K130 with the aromatic residue F134 contributes to steric constraints over the C(19) methyl pyridine group (T stacking), an orientation of the keto group towards K130 and the methoxy group positioned in the polar area This polar area is formed by H128, Q218 [hydrogen bond to keto group of androstenedione (C17)], Q225 (electrostatic interactions with D476), H475 and D476 It has been previously shown that mutation at position Q225 modified the Km.app of the enzyme [24] The impact of mutations on the IC50 value is consistent with the hypothesis Indeed the mutations D476N, D476A and H475A not only decrease the polar characteristic of the region called Ôpolar areaÕ that support the interaction with the methoxy group of the inhibitor for MR20814, but also the hydrophobic interaction with the pyrrole group for the two other inhibitors (MR20492, MR20494) The D476E mutation could stabilize the position of the ligand by an electrostatic interaction with the amino group of MR20814 The K130N mutation avoided the electrostatic repulsion between the amino group of MR20814 and K130, allowing a better position of the ligand inside the active site (conformational flexibility) Ó FEBS 2002 Structure–function relationships of human aromatase (Eur J Biochem 269) 1401 Fig Hypothesis on the position of MR20814 inside the active site The position of MR20814 inside the active site correspond to: an orientation of the amino group toward the hydrophobic area composed of residues I474 and L477, an interaction between the pyridine group and F134, an orientation of the keto group toward K130 and the methoxy group positioned in the polar area H128, Q218, Q225, H475 and D476 form this polar area H475 Mutation of H475 of hP-450arom to Asn lowered the activity by 95%, suggesting that this residue is important at this position Graham-Lorence et al [20] suggested that H475, or K473, could facilitate the enolization of the 3-keto group of the substrate In order to define the role of this residue, we transfected the mutants H475R, H475E and H475A According to our molecular model, H475 is closer to O(3) of the substrate, compared to K473 but H475 seemed to be an unlikely candidate for proton donation to the substrate 3-keto group since H475R was slightly active, whereas H475A remained fully active However, a hydrophobic residue seemed to be crucial at this position as observed with H475N, H475E and H475R, which were weakly active H475, located in a polar area and close to the hydrophobic cluster which begins and ends with I474 and L477, by its hydrophobic characteristic would then have a role in the stabilization of the substrate in the active site Indeed, only H475A was active and had a 2.5-fold increased binding affinity for androstenedione, suggesting a modification of the hydrophobic properties of the cluster This reinforces also the inhibitory potencies of the three molecules tested I474 This position was extensively studied [21,53] in order to evaluate the extra hydrophobic pocket of the enzyme According to Kao et al [21], I474 would be on an extrahydrophobic surface rather than in a hydrophobic pocket, as described by Graham-Lorence et al [20], based on the fact that mutation of I474 to bulky and hydrophobic aromatic residues (I474Y, I474W) maintained the potency of the 7a-APTA inhibitor Similar results were obtained by Zhou et al [53] with mutant I474F In our study, the mutant I474T presented a weak binding affinity for androstenedione, but a Vm.app value similar to that of the wild-type aromatase, and it was less inhibited by the nonsteroidal inhibitors known previously to interact with the hydrophobic surface [11–13] Presence of a polar residue Fig View of the hydrophobic surface (MOLCAD SURFACE) The definition of the lipophilicity potential made it possible to define the hydrophobic surface (brown), the neutral surface (green) and the hydrophilic surface (blue) The position of MR20814 inside the active site corresponds to an orientation of its amino group toward the hydrophobic surface, previously described as the extrahydrophobic surface (21), compared of residue I474 and L477 on this hydrophobic surface of the active site could reduce desolvation effects leading to a decreasing binding of the substrates and inhibitors (MR 20814, MR 20492 and MR 20494) This hypothesis is supported by the observation that a previously described mutant I474N showed decreasing binding affinity for androstenedione whereas mutations with hydrophobic residues (I474F, I474W, Y474Y, I474M) did not [21,53] Furthermore, results obtained for the I474T mutant, with those obtained for H475N, suggest that this face of the active site has a slightly reduced hydrophobic interaction with the substrate for the equine enzyme compared with the human counterpart This could be also the case for the porcine isoforms (K474 and N475) F320 and S470 These residues are not located in the substrate-binding site but their mutations could indirectly influence its structure With regard to the F320 residue, positioned in the helix I, the results indicated that its mutation to Cys had weakly increased binding affinity for androstenedione and decreased Vm.app value for nortestosterone The F320 residue is bulky and seems to be ˚ close to S470 (4.5 A) The F320C mutation could lead to the ˚ creation of a disulfide bond between C320 and C467 (3.5 A) as was previously proposed [11] This new bond may then modify the loop structure S470–S478 leading to a modification of the polar area (H475 and D476) and the hydrophobic surface (L477, I474) explaining the impact on the affinity of the substrates, particularly testosterone, and of the inhibitors by disfavoring the interaction of the pyridyl group with the heme This could be explained by the impact of the position of an acidic group towards the affinity of testosterone (D476E and this result) These results also confirm the different position of testosterone and nortestosterone in the active site (see Discussion on enzymatic mechanism) S470N (polar residue) mutation showed also a variation in Km.app value for androstenedione and a decrease Ó FEBS 2002 1402 P Auvray et al (Eur J Biochem 269) in Vm.app value for nor-testosterone These effects, similar to those observed for F320C, led to the hypothesis that this mutation has an impact on the loop structure Residues toward the methyl groups (b-face) of the steroid – K130 According to the theoretical model of GrahamLorence et al [20], K130 could be involved in a salt-bridge with E302, this possibly aiding reprotonation of D309 and T310 Furthermore, if the amino group of K130 pairs with E302, its long aliphatic lateral chain will reinforce hydrophobic interactions, controlled also by F134, with the b-face of the steroid The role of K130 in reprotonation was not confirmed, as the mutation of K130 to Asn had no effect on the catalytic properties of the enzyme with androstenedione as a substrate According to our molecular model, reprotonation of D309 could be inferred from the conserved residue K473, which is closer to D309 than to the 3-keto group of the substrate Another possibility is that the amino group of K130 strongly interacts with the aromatic residue F134 (quadrupole–quadrupole interactions) rather than with E302, and this complex contributes to steric constraints over the C(19) methyl group of androgens However, K130N showed 70% decrease in the Km.app value using testosterone as a substrate, but there was a twofold increase of the Km.app value with nor-testosterone as a substrate, and the Vm.app was 36% and 18% that of the wild-type for testosterone and nor-testosterone, respectively These effects on substrate binding could be explained by taking into account the roles of several residues: (a) the stabilizing role of the polar area towards the polar group on C17 (keto or hydroxyl group); (b) the impact of an acidic group on the 17-hydroxyl of testosterone and nor-testosterone (D476E); (c) the steric constraints of the K130-F134 complex to the C(19) methyl of testosterone; and (d) the role of D309 and of E302 in aromatization of androgens and nor-androgens, respectively Thus, N130, located at the opposite face of D476, could create a second polar area (Fig 8) formed by H128 and N130, leading to new electrostatic interactions with the 17-hydroxyl group of testosterone and of nortestosterone Consequently, the holding of the C19 methyl near the heme iron could be affected, which would provide an explanation to the lower Km.app and Vm.app values observed for testosterone Contrasting with the results obtained with testosterone, the K130N mutation largely decreased the binding affinity for nor-testosterone, suggesting that this residue, along with F134, may have a more important role on substrate binding of nor-androgens as suggested previously The 80% drop of the Vm.app value for nor-testosterone may result from the wrong holding of ring A near the heme iron and near E302 Residues toward the D-ring of the steroid – K119 Laughton et al [25] suggested that this residue would interact with the heme carboxylate group whereas GrahamLorence et al [20] indicated that K119 was on the external surface of the protein and orientated outwards In our molecular model, K119 was also pointing towards the solvent and located in a short b sheet between two glycines involved in loop structures (GSKLG) External ionic residues, such as glutamic acid, could maintain this structure, whereas hydroxylated residues, such as threonine and tyrosine, will modify it The other possibility is that K119 is not outside the protein surface but rather inside, as Fig Representation of the position of this new polar area towards the last polar area N130 located at the opposite face of D476 could create a second polar area, with H128, leading to new electrostatic interactions with the C(17) group of the steroid our mutants modified the binding affinity for androstenedione and the inhibitory potential of inhibitors In this case, by introducing a hydroxyl group, accessibility of the substrate to the active site is reduced, as observed with the mutants K119T and K119Y By keeping an ionic residue, the binding affinity for androstenedione and the inhibitory potential of MR 20492 are increased as observed for the K119E mutant This residue appears to be significant and may be involved indirectly in the structure of the L122– H128 domain interacting with the D-ring of the steroid as suggested by the catalytic differences of K119E observed with androstenedione or testosterone as substrates I125 According to our molecular model, this residue is in the L122–H128 domain of the active site, which has been implicated in an extrahydrophobic pocket surrounding the D-ring of the steroid and the polar area (H128) Interestingly, I125M, which was not susceptible to modify the hydrophobic surface, had no activity These results may be explained by the possibility of a strong interaction of the ˚ methionine with the aromatic residue F221 (3 A) This interaction could increase the steric constraints over the D-ring of the steroid and could be involved in the lower efficiency of the 16a-hydrotestosterone aromatization by the equine enzyme than by the human one [63] Differences between the two enzymes was also apparent in the C124Y mutant suggesting that an increase of the hydrophobic character of this part of the human active site modified the aromatization efficiency of androstenedione but increased the inhibition potency of MR 20814 and MR 20492 CONCLUSIONS In this study, we explored the role of residues within the active site of the human more specifically those involved in substrate interactions and catalysis Our results indicate that some mutants show variable activity with androstenedione, testosterone or nor-testosterone These results suggested that the residues involved in substrate stabilization may vary, or had differing importance depending on the Ó FEBS 2002 Structure–function relationships of human aromatase (Eur J Biochem 269) 1403 substrate For instance, we highlight the importance of the L122–H128 domain (results obtained with the K119, I124, I125 mutants) of the loop structure corresponding to S470– S478 (results obtained with the S470, I471, I474, H475, D476 mutants) in the stabilization of the steroid (D-ring) and particularly the role of D476 Positioning of the steroid inside the active site will differ according to its C(17) group and modification of the polarity of these two domains (L122–H128 and S470–S478) in different species Furthermore, residues K130 and F134 appear to form a hydrophobic surface close to the heme and control the position of the substrate inside the active site (differences for norandrogens and androgens) These results are the first report using nor-testosterone as substrate for aromatase mutants They suggest that E302 interacts with C(2) of nor-androgens and is implicated in their aromatization, whereas decarboxylation and aromatization of androgens involve D309 Most of the substitutions of human residues by their corresponding aligned equine residues, conservatives or not, produced differences in the catalytic properties of the enzyme suggesting that incompletely conserved residues may be involved in biochemical specificities between species, including differences in inhibition This is also supported by the recent work of Corbin et al [27] and Kao et al [28], which demonstrate catalytic differences between porcine isoforms of aromatase The main differences, which could account for differences in biochemical specificities between equine and human enzymes, are residues N475, N130 and M125 Such comparative studies of aromatase across species has shed new light on the aromatization mechanism and the structure of the active site ACKNOWLEDGEMENTS This work was initiated and developed through a grant from the Ligue ´ Nationale Contre Le Cancer (Comite de la Manche et du Calvados) for the biological studies, and a studentship to P A., and through a grant ´ from the Region de Basse-Normandie and, FEDER (Comite Agrobio) We would also like to thank Prof W.R Miller (Edinburgh, UK) and C Joubel 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directed mutagenesis study Eur J Biochem 268, 243–251 63 Gaillard, J.L (1991) Equine testicular aromatase: substrates specificity and kinetic... the study of their catalytic differences [27,28] also highlight the need to understand which residues of the enzyme are involved in the substrate specificity In addition to human and porcine aromatases,