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
Trang 1Study of substrate specificity of human aromatase by site directed mutagenesis
P Auvray1,*,†, C Nativelle1,†, R Bureau2, P Dallemagne2, G.-E Se´ralini1and P Sourdaine1
1
IBBA, Laboratoire de Biochimie et Biologie Mole´culaire, Universite´ de Caen, Esplanade de la Paix, Caen, France;
2
CERMN, Laboratoire de Pharmacochimie, Caen cedex, France
Human aromatase is responsible for estrogen biosynthesis
and is implicated, in particular, in reproduction and
estro-gen-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,
testoster-one or nor-testostertestoster-one as substrates In addition, inhibition
profiles for these mutants with indane or indolizinone
derivatives were obtained Our results, together with
com-puter modeling, show that catalytic properties of mutants
vary in accordance with the substrate used, suggesting possible differences in substrates positioning within the act-ive site In this respect, importance of residues H475, D476 and K130 was discussed These results allow us to hypo-thesize 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 conforma-tion, and thus sheds new light on residues that may be responsible for substrate specificity between species or aro-matase isoforms
Keywords: aromatase; site-directed mutagenesis; molecular modeling; androgens; inhibitors
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
cyto-chrome P-450 aromatase and the ubiquitous cytocyto-chrome
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 cyto-chromes Pseudomons putida camphor P-450 (P-450cam), Bacillus megaterium P-450 (P-450BM-3), Pseudomonas putidaa-terpineol P-450 (P-450terp) and Saccharopolyspora erythreaeerythromycin 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-dimen-sional structures, have very weak sequence homologies with P-450arom Moreover, FASTA3AND BLAST analyses of the protein databank sequences do 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 2 31 56 53 20,
Tel.: + 33 2 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)
Trang 2a 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
aroma-tase, 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]
E X P E R I M E N T A L P R O C E D U R E S
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
from Dupont NEN (Les Ulis, France), testosterone and 19-nor-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, Que´bec), Quick-ChangeTMSite-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 construc-tion were checked by 1% agarose electrophoresis and ethidium bromide staining
Fig 1 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].
Fig 2 Structure of inhibitors 4-OHA is from Brodie et al [64], MR
20814, MR 20492 and MR 20494 from Auvray et al [11,13].
Trang 3Aromatase cytochrome P-450 molecular modeling
Initial alignments of cytochrome P-450 BM3 and
aroma-tase 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
usingAMBER[36] andESFF FORCE FIELD(ProgramDISCOVER
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 kcalÆmol)1ÆA˚)1, then
conjugate gradients until a gradient of 1 kcalÆmol)1ÆA˚)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
orien-tation of C(1), C(2) and C(19) above the heme [41] and the
position of the ligand towards D309 and T310 The
three-dimensional 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
ISOSTARsoftware [43] Theoretical noncovalent interactions
from theISOSTARsoftware 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 disper-sion energy (calculated at the second order double excitation level)
The definition of the lipophilicity potential (MOLCAD SURFACE; programSYBYL6.0; 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 Site-Directed Mutagenesis method from Stratagene Briefly, this was based on a PCR with two complementary oligonucle-otide 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 min 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 coliXL1-Blue supercompetent cells Transformed bacteria were analyzed directly on colonies by PCR with primers
5H-1 (5H-1365H-1–5H-1379, 5¢-GTCGTGTCATGCTGGACAC-3¢) and 3H-54 (2384–2367, 5¢-GAGGATGACACTATTGGC-3¢) after 30 cycles (1 min at 95°C, 1 min at 52 °C, 2 min at
72°C; 1 cycle: 10 min at 72 °C) The expected amplicon length was 1026 bp Mutations were then checked by sequencing 10 lL bacterial DNA miniprep with the
Ther-mo Sequenase, as previously described [13] Plasmid DNA was extracted as follows: the bacterial pellet was lysed with 8% sucrose, 0.5% Triton X-100, 0.05M EDTA, 0.01M Tris/HCl pH 8.0 and 10 mgÆmL)1 lysosyme/0.01M 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 supercompe-tent 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 2 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 2 lg pCMV-human aromatase cDNA, using a modification of the method of Boussif et al [46] Briefly, 2 lg pCMV-cDNA (6 nmol of phosphate) and 54 nmol of polyethylen-imine were separately diluted with 50 lL 150 mM NaCl, incubated for 10 min at room temperature in a laminar fume hood, mixed together, incubated for another 10 min at room temperature, and then added to each well Cells were incubated for 3–4 h at 37°C and then supplemented with
Trang 4500 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
micro-somal 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
3H2O released from [1b,2b-3H]-androstenedione Cells were
washed with serum-free culture medium Dessicated
radio-active substrate (200 nMand 50–800 nMfor IC50and kinetic
experiments, respectively) supplemented with 1 lM
prog-esterone (used to block 5a-reductase activity) and 0–10 lM
inhibitor (for IC50experiments) were mixed with serum-free
culture medium and added to each well Cells were
incubated at 37°C under 5% CO2 for 45 min After
incubating cells for 5 min on ice, the culture medium
(1 mL) was sampled and extracted by CHCl3 (1 mL)
Steroids were then removed by incubation with 1 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
pmolÆmin)1Æmg aromatase)1 Results from control
incuba-tions, 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 min (nor-testosterone) or 90 min
(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, bioMe´rieux, Charbonnie`res les Bains, France] were
diluted twofold according to the manufacturer’s
instruc-tions The extraction efficiency was 80 ± 5% and the
sensitivity of this radioimmunoassay was 10 pgÆmL)1
Results, calculated according to Garnier et al [49], were
the mean of at least triplicate experiments ± SD and are
expressed as pmolÆmin)1Æmg 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
(1 : 10 000), raised against intact eP-450arom [29], incuba-ted for 2 h and then added (100 lL per well) to plates (Nunc, high protein adsorption quality) Plates were previ-ously coated overnight at 4°C with 50 ng per well of purified equine aromatase, saturated 1 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 1 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)
R E S U L T S
The catalytic properties of P450arom mutants are summar-ized in Tables 1 and 2 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 2 IC50 are presented in Table 3 and are analyzed in respect of catalytic properties of mutants with the substrates tested
Western blot analysis [29] demonstrated that the poly-clonal antibodies specifically detected human aromatase in microsomes Aromatase was also evident in E293 cells
Fig 3 Standard curve of ELISA (A) The standard curve was obtained by mixing 200 lL NaCl/P i containing 0–8 ng of purified equine aromatase to 800 lL of polyclonal anti-(ep.450arom) Ig;
1 : 10 000 The fixation of the primary antibody was then evaluated with anti-(rabbit IgG) Ig coupled to alkaline phosphatase and incu-bation 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 2 lg pCMV-human aromatase cDNA The size and position of the expected 55 kDa aromatase protein is shown.
Trang 5transfected 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, n¼ 305), 2.33 ± 0.9 ng
for the wild type (n¼ 68) and with a range of
2.11 ± 0.6 ng for D476A (n¼ 5) to 4.48 ± 2.63 ng for
K130N (n¼ 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
de-creased aromatase activity: H475N, H475R, H475E,
D476A, D476K and D476L (activities below 1 or 5%
relative to wild-type with 200 nM of androstenedione,
Table 1) In contrast, the Km.appvalue for H475A and the
Vm.appvalue 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
nor-testosterone as substrates (Table 2) The decrease of the
binding affinity of testosterone for mutant D476N was
accompanied by a decrease in the V value although
catalytic properties of D476N were unchanged with nor-testosterone A lower aromatase activity with these sub-strates was observed for D476E (13% and 8% of wild-type for testosterone and nor-testosterone, respectively), con-trasting with the results obtained with androstenedione The relative potency of the three inhibitors tested, accord-ing to their IC50 values (Table 3), was increased for inhibition of H475A, which had a lower Km.appvalue 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 decarboxy-lation 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 Further-more, E302A was inactive with all substrates tested Human residues implicated in the active site of the aromatase were mutated to corresponding aligned equine
Table 1 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- 3 H]-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 n M of androstenedione corresponding to an activity of 1497 ± 300 pmolÆmin)1Æmg aromatase)1).
Protein K m.app (n M ) Wild-type K m (%) V m.app (pmolÆmin)1Æmg)1) Wild-type V m (%)
a
P < 0.05.bP < 0.005.cP < 0.001 ( ANOVA ).
Trang 6residues The mutant K130N had similar catalytic
proper-ties for androstenedione when compared to the wild-type
enzyme (Table 1) K119T also had greatly reduced
aroma-tase activity as was also observed for C124Y (but to a lesser
extent), which had a lower Vm.appvalue 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.appvalue
for androstenedione Interestingly, the binding affinity of
K119E for testosterone was unchanged when compared to the one of wild-type P450arom but the Vm.appdecreased 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.appvalue were lower for nor-testoster-one The mutant F320C, weakly active with testosterone, was found to have a lower Vm.appvalue 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 1 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.appvalues 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
D I S C U S S I O N
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 docu-mented model of Graham-Lorence et al [20] together with results from our molecular model
The Km.appof the recombinant wild-type aromatase for androstenedione (164 ± 38 nM) is in the range of Kmvalues reported in the literature (9–150 nM) [14,21,24,52–54] In our study, the Km.appfor testosterone is slightly higher than for androstenedione, as was observed by other groups
Table 2 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 n M of testosterone corresponding to an activity of 1731 ± 180 pmolÆmin)1Æmg arom)1 or with 200 n M of nor-testosterone corresponding to an activity of 2287 ± 477 pmolÆmin)1Æmg arom)1).
Protein
K m.app (n M ) V m.app (pmolÆmin)1Æmg)1) K m.app (n M ) V m.app (pmolÆmin)1Æmg)1)
a
P < 0.05 ( ANOVA ).
Table 3 IC 50 values with steroidal and nonsteroidal aromatase
inhibi-tors used with human aromatase mutants Aromatase activity was
evaluated by measuring the amount of 3 H 2 O released from 200 n M
[1b,2b-3H]-androstenedione incubated in culture medium at 37 °C-5%
CO 2 atmosphere for 45 min in presence of inhibitors Aromatase
ac-tivities were expressed as percentage of a standard control which was
incubated without inhibitor in the same conditions IC 50 values in l M ,
were the mean ± SD of three (wild-type) or two (mutants) experiments
in triplicate IC 50 value with 4OHA, used as control, was
0.45 ± 0.35 l M with the wild-type protein.
Protein
IC 50 (l M )
Wild-type 10.83 ± 0.80 3.93 ± 0.93 0.23 ± 0.02
K119Y 3.76 ± 1.68a 5.72 ± 1.10 0.17 ± 0.03
K119V > 10 6.21 ± 2.60 1.55 ± 0.5a
K119E 9.80 ± 0.42 0.74 ± 0.17 a 0.53 ± 0.45
C124Y 4.40 ± 1.97a 0.33 ± 0.25a 0.33 ± 0.18
K130N 0.23 ± 0.16a 4.27 ± 2.58 0.20 ± 0.13
F320C > 10 5.35 ± 1.12 0.94 ± 0.50 a
H475A 0.92 ± 0.12a 0.45 ± 0.007a 0.10 ± 0.00a
D476N 2.02 ± 1.44 a 0.92 ± 0.12 a 0.15 ± 0.07
D476E 4.68 ± 0.12 a 5.60 ± 0.28 a 0.15 ± 0.07
a
P < 0.05 ( ANOVA ).
Trang 7[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.appvalue for
nor-testoster-one when compared to the value obtained for testosternor-testoster-one
These results are in accordance with those obtained by
Kellis & Vickery [55] for nor-androstenedione and
andros-tenedione
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 Vmobtained in this
study with those reported in the literature is more difficult
By using ELISA, we found aromatase in human placental
microsomes at 210 pmolÆmg 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.appbetween 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)
(aromatiza-tion), 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 con-straints, 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 corres-ponding 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]
Fig 4 Enzymatic mechanism of the human
aromatase with androgens (A) and
nor-andro-gens (B) The enzymatic mechanism was
modified from Graham-Lorence [20] and from
Ahmed [41,59].
Trang 8In their recent study, Kao et al [62] emphasized the
importance of the interaction between E302 and
andros-tenedione 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
aroma-tase 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.appand
Vm.appvalues with androstenedione However, with
testos-terone and nortestostestos-terone, 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)1withISOSTARsoftware), could destabilize the
position of these ligands inside the active site
When compared to the wild-type enzyme, D476N had
lower Vm.appvalues for C19-androgens but similar Km.app
and Vm.appvalues 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 6
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
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.appof the enzyme [24]
The impact of mutations on the IC50value is consistent with the hypothesis Indeed the mutations D476N, D476A and H475A not only decrease the polar characteristic of the region called Ơpolar 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)
Fig 5 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.
Trang 9H475 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.appvalue 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
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 nor-testosterone 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 modifi-cation 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 nor-testosterone in the active site (see Discussion on enzymatic mechanism) S470N (polar residue) mutation showed also a variation in K value for androstenedione and a decrease
Fig 7 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.
Fig 6 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
com-posed 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.
Trang 10in Vm.appvalue 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
Graham-Lorence 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
hydro-phobic 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,
repro-tonation 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.appvalue 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
nor-testosterone 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,
suggest-ing 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.appvalue 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
Graham-Lorence 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
our mutants modified the binding affinity for androsten-edione 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 theD-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) Interest-ingly, 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
C O N C L U S I O N S
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
Fig 8 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 interac-tions with the C(17) group of the steroid.