Eva Stjernschantz

Jelle Reinen

Walter Meinl

Beena J George

Hansruedi Glatt

Nico P E Vermeulen

Chris Oostenbrink
email: c.oostenbrink@few.vu.nl

Comparison of murine and human estrogen sulfotransferase inhibition in vitro and in silico-implications for differences in activity, subunit dimerization and substrate inhibition

Keywords: estrogen sulfotransferase, substrate inhibition, molecular dynamics, simulation ED, endocrine disruption, EDC, endocrine disrupting compound, SULT1E1, sulfotransferase

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Introduction

The presence of estrogens in the environment and their possible role in the process of endocrine disruption (ED) has been the subject of a large number of studies in the past years [START_REF] Bolger | Rapid screening of environmental chemicals for estrogen receptor binding capacity[END_REF][START_REF] Harris | Phytoestrogens are potent inhibitors of estrogen sulfation: implications for breast cancer risk and treatment[END_REF][START_REF] Hotchkiss | Fifteen years after "Wingspread"-environmental endocrine disrupters and human and wildlife health: where we are today and where we need to go[END_REF]. Endocrine disrupting compounds (EDCs) can interfere with the endocrine system by either mimicking or preventing the action of physiological hormones. They can also act by altering the synthesis and function of hormone receptors and by modifying the synthesis, transport, metabolism and excretion of hormones [START_REF] Ropero | Rapid endocrine disruption: environmental estrogen actions triggered outside the nucleus[END_REF].

Estrogen sulfotransferase (SULT1E1) is an important enzyme involved in the metabolism of 17β-estradiol (E2), the natural ligand of the mammalian estrogen receptors (ERs). This cytosolic sulfotransferase catalyzes the transfer of a sulfuryl group (SO 3 -) from the ubiquitous donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to E2 and other estrogens at the 3-hydroxy position. The resulting estrogen sulfates are highly water-soluble and unable to bind to the ER; instead they are either excreted from the cell or stored for reactivation by sulfatases [START_REF] Strott | Steroid sulfotransferases[END_REF][START_REF] Song | Steroid transformation enzymes as critical regulators of steroid action in vivo[END_REF]Negishi 2001). SULT1E1 shows high affinity for E2 and estrone (E1) and is involved in the regulation of E2 responsiveness at physiological concentrations [START_REF] Zhang | ), S = substrate concentration. S(mSULT1E1) = 250 nM , S(hSULT1E1) = 18.75 nM. K m (mSULT1E1) = 305 nM, K m (hSULT1E1) = 6.4 nM. c Ratio is defined as K i[END_REF][START_REF] Glatt | Pharmacogenetics of soluble sulfotransferases (SULTs)[END_REF]. It is believed that SULT1E1 plays an important role in protecting peripheral tissues from excessive estrogenic effects. Increased local availability of biologically active estrogens caused by decreased SULT1E1 expression or SULT1E1 inhibition may be related to various estrogen-dependent properties, such as the development and maintenance of hormone-dependent breast carcinomas [START_REF] Qian | Expression of estrogen sulfotransferase in MCF-7 cells by cDNA transfection suppresses the estrogen response: potential role of the enzyme in regulating estrogen-dependent growth of breast epithelial cells[END_REF][START_REF] Suzuki | Steroid sulfatase and estrogen sulfotransferase in normal human tissue and breast carcinoma[END_REF][START_REF] Utsunomiya | Steroid sulfatase and estrogen sulfotransferase in human endometrial carcinoma[END_REF]). Due to the critical role in controlling local estrogen levels, it is The function and reaction mechanism of cytosolic sulfotransferases have been studied

extensively through structural as well as mutational and kinetic studies [START_REF] Kakuta | mechanism. Crystal structure of a vanadate complex of estrogen sulfotransferase and mutational analysis[END_REF]Kakuta 1998b;[START_REF] Zhang | ), S = substrate concentration. S(mSULT1E1) = 250 nM , S(hSULT1E1) = 18.75 nM. K m (mSULT1E1) = 305 nM, K m (hSULT1E1) = 6.4 nM. c Ratio is defined as K i[END_REF][START_REF] Pedersen | Crystal structure of the human estrogen sulfotransferase-PAPS complex: evidence for catalytic role of Ser137 in the sulfuryl transfer reaction[END_REF][START_REF] Hoff | Transition state of the sulfyryl transfer reaction of estrogen sulfotransferase[END_REF][START_REF] Tyapochkin | Isotope exchange at equilibrium indicates a steady state ordered kinetic mechanism for human sulfotransferase[END_REF]). The reaction mechanism has been proposed to be of a sequential kinetic nature, involving the formation of a ternary enzyme complex before the products are released. This sequential mechanism can either proceed in an ordered bi-bi fashion, where the cofactor and the substrate bind in an ordered sequence, or through a random bi-bi mechanism, as displayed in Figure 1. Both random and ordered sequential mechanisms can lead to dead-end complexes and both mechanisms have been proposed for cytosolic sulfotransferases [START_REF] Zhang | ), S = substrate concentration. S(mSULT1E1) = 250 nM , S(hSULT1E1) = 18.75 nM. K m (mSULT1E1) = 305 nM, K m (hSULT1E1) = 6.4 nM. c Ratio is defined as K i[END_REF][START_REF] Chapman | Sulfotransferases: structure, mechanism, biological activity, inhibition, and synthetic utility[END_REF][START_REF] Gamage | The structure of human SULT1A1 crystallized with estradiol. An insight into active site plasticity and substrate inhibition with mult-ring substrates[END_REF][START_REF] Hoff | Transition state of the sulfyryl transfer reaction of estrogen sulfotransferase[END_REF][START_REF] Allali-Hassani | Structural and chemical profiling of the human cytosolic sulfotransferases[END_REF].

The sulfuryl transfer reaction has been proposed to proceed through an inline, S N 2-like displacement mechanism, involving a nucleophilic attack on the cofactor sulfonate by the substrate (Kakuta 1998b). A highly conserved histidine residue (His107 in hSULT1E1) is believed to deprotonate the hydroxyl group of the substrate, which can subsequently attack the sulfur atom of the cofactor, as displayed in Figure 2. In addition to His107, studies indicate two other residues important for catalysis [START_REF] Kakuta | mechanism. Crystal structure of a vanadate complex of estrogen sulfotransferase and mutational analysis[END_REF]Kakuta 1998b). Lys105

positions the substrate close to the cofactor and interacts with the sulfonate, thereby stabilizing the transition state. Lys47 seems to stabilize the transition state through donating a proton to the bridging oxygen of the cofactor, acting as a catalytic acid, and interacts with one of the sulfonate oxygens. His107 also seems to serve a stabilizing role through interaction with another of the sulfonate oxygens [START_REF] Kakuta | mechanism. Crystal structure of a vanadate complex of estrogen sulfotransferase and mutational analysis[END_REF]Kakuta 1998b). Until recently all crystal A c c e p t e d M a n u s c r i p t co-crystallized with different substrates. Earlier this year, Teramoto et al published a crystal structure of mouse sulfotransferase SULT1D1 in complex with both PAPS and substrate. This structure confirms the results of the previous studies [START_REF] Teramoto | Snapshot of a Michaelis complex in a sulfuryl transfer reaction: Crystal structure of a mouse sulfotransferase, mSULT1D1, complexed with donor substrate and acceptor substrate[END_REF].

Several studies have shown that hSULT1E1 and other members of the sulfotransferase family exhibit substrate inhibition at higher substrate concentrations, as well as noncompetitive binding of inhibitors (Falany 1995;[START_REF] Kester | Potent inhibition of estrogen sulfotransferase by hydroxylated PCB metabolites: a novel pathway for explaining the estrogenic activity of PCBs[END_REF][START_REF] Kester | Potent inhibition of estrogen sulfotransferase by hydroxylated metabolites of polyhalogenated aromatic hydrocarbons reveals alternative mechanism for estrogenic activity of endocrine disruptors[END_REF][START_REF] Tyapochkin | Isotope exchange at equilibrium indicates a steady state ordered kinetic mechanism for human sulfotransferase[END_REF].

Other reports suggest non-productive substrate binding modes or binding of multiple substrates in the active site of different sulfotransferases [START_REF] Rehse | Crystal structure of human dehydroepiandrosterone sulphotransferase in complex with substrate[END_REF][START_REF] Gamage | Structure of human carcinogen-converting enzyme SULT1A1. Structural and kinetic implications of substrate inhibition[END_REF][START_REF] Lee | Crystal structure of human cholesterol sulfotransferase (SULT2B1b) in the presence of pregnenolone and 3'-phosphoadenosin 5'-phosphate[END_REF]Barnett 2004). There has been considerable debate in the literature whether these observations originate from the presence of a second, allosteric, binding site or can be explained by the formation of a dead-end complex through the binding of substrates or inhibitors to the enzyme with bound inactive cofactor, PAP [START_REF] Zhang | ), S = substrate concentration. S(mSULT1E1) = 250 nM , S(hSULT1E1) = 18.75 nM. K m (mSULT1E1) = 305 nM, K m (hSULT1E1) = 6.4 nM. c Ratio is defined as K i[END_REF]Cui 2004;[START_REF] Gamage | The structure of human SULT1A1 crystallized with estradiol. An insight into active site plasticity and substrate inhibition with mult-ring substrates[END_REF]. No allosteric site outside the active site has been identified in any of the published crystal structures. However, most of the substrate-containing structures result from crystal soaking experiments, which may prevent the binding of the substrate to an external allosteric site, if large conformational changes are required [START_REF] Shevtsov | Crystallographic analysis of a hydroxylated polychlorinated biphenyl (OH-PCB) bound to the catalytic estrogen binding site of human estrogen sulfotransferase[END_REF]. Zhang et al suggested that in the dimeric hSULT1E1, one of the active sites is the catalytic site, whereas the other serves as an allosteric site, regulating catalysis through substrate inhibition [START_REF] Zhang | ), S = substrate concentration. S(mSULT1E1) = 250 nM , S(hSULT1E1) = 18.75 nM. K m (mSULT1E1) = 305 nM, K m (hSULT1E1) = 6.4 nM. c Ratio is defined as K i[END_REF]). Gamage et al proposed that a second molecule in the active site could be the reason for substrate inhibition of human SULT1A1 at high substrate concentrations [START_REF] Gamage | Structure of human carcinogen-converting enzyme SULT1A1. Structural and kinetic implications of substrate inhibition[END_REF].

Alternatively, substrate binding in a non-productive binding mode in the active site at high concentrations could be the origin of substrate inhibition [START_REF] Gamage | The structure of human SULT1A1 crystallized with estradiol. An insight into active site plasticity and substrate inhibition with mult-ring substrates[END_REF].

A c c e p t e d M a n u s c r i p t 6 Unlike most sulfotransferases, mSULT1E1 does not seem to display substrate inhibition to the same extent as the human enzyme [START_REF] Falany | Enzymology of human cytosolic sulfotransferases[END_REF]Kakuta 1998a;Qian 1999).

The crystal structures of human and murine SULT1E1 bound to their natural substrate E2

have been published and show high similarity both in structure and mode of substrate binding [START_REF] Pedersen | Crystal structure of the human estrogen sulfotransferase-PAPS complex: evidence for catalytic role of Ser137 in the sulfuryl transfer reaction[END_REF]. A significant difference between the murine and human enzyme is that the murine enzyme is biologically active as a monomer, whereas the human one only functions as a dimer (Petrotchenko 2001). It is therefore interesting to study whether the apparent differences between the human and murine SULT1E1, in terms of substrate inhibition and differences in biologically active forms of the proteins, correlate with a functional difference between human and murine SULT1E1 and what the origin of this possible functional difference is.

We have recently developed a fluorescence HPLC-based screening assay for inhibition of hSULT1E1 [START_REF] Reinen | Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase[END_REF]) which makes use of the nonradioactive and non-carcinogenic substrate 1-hydroxypyrene (OHP) and hSULT1E1 expressed in Salmonella typhimurium.

Here we compare the enzymatic and inhibitory properties of human and murine SULT1E1 in vitro and in silico.

Cytosolic preparations

The cDNA of mouse SULT1E1 (mSULT1E1; GenBank accession no. NM 023135)

was cloned and expressed in Escherichia coli XL-1 Blue cells using the pKK233-2 expression

A c c e p t e d M a n u s c r i p t 8 vector as previously described for the various human sulfotransferases (Falany 1995). The plasmid was adapted to the restriction enzymes of Salmonella typhimurium LT2 by passing it through the restriction-deficient, but methylation-proficient Salmonella typhimurium strain LB5000. The plasmid was then used to transform the his -strain TA1538. The newly generated Salmonella typhimurium strain was designated TA1538-mSULT1E1. mSULT1E1 constituted approximately 5% of the total cytosolic protein.

The transformed bacterial strain was grown overnight in the presence of ampicillin (100 µg/ml) to reinforce the maintenance of the recombinant plasmid. Bacterial cytosol was prepared by ultrasonication and dialyzed for 4 h against a 100-fold excess of buffer (150 mM KCl in 10 mM sodium phosphate buffer (pH 7.4)) (Glatt 1995a;Glatt 1995b).

Human SULT1E1 (hSULT1E1) and SULT1A1 (hSULT1A1) were expressed as described previously [START_REF] Reinen | Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase[END_REF]. Bacterial cytosol was prepared as described above. The use of Salmonella cytosol has advantages over the use of purified SULTs. Stabilization was observed for various SULTs (e.g. SULT1A1) in the presence of the cytosolic proteins.

Moreover, Salmonella cytosol rapidly degrades PAP (H. R. Glatt, unpublished result), the product formed from PAPS during the sulfuryl transfer reaction, which is a potent inhibitor of various SULTs, including human SULT1A1 (Rens-Domiano 1987) and hSULT1E1 [START_REF] Zhang | ), S = substrate concentration. S(mSULT1E1) = 250 nM , S(hSULT1E1) = 18.75 nM. K m (mSULT1E1) = 305 nM, K m (hSULT1E1) = 6.4 nM. c Ratio is defined as K i[END_REF].

HPLC analysis of 1-hydroxypyrene and pyrene 1-sulfate

In order to measure the sulfonation of 1-hydroxypyrene, HPLC analysis was performed as reported previously [START_REF] Reinen | Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase[END_REF] Since no chemically prepared pyrene 1-sulfate was available, calibration curves of pyrene 1-sulfate were made as described previously [START_REF] Reinen | Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase[END_REF]). Briefly, OHP was incubated in the presence of PAPS and hSULT1A1 with a resulting pyrene 1-sulfate yield of more than 97% in all cases [START_REF] Ma | Solvent effect on cDNA-expressed human sulfotransferase (SULT) activities in vitro[END_REF]. These calibration curves were made for each single enzyme kinetic experiment to accurately determine the amounts of pyrene 1-sulfate formed using the HPLC analysis procedure described above.

Protein and time dependency and enzyme kinetics of murine SULT1E1-mediated pyrene 1-sulfate formation

Linearity of the protein-dependent formation of pyrene 1-sulfate and its time dependency were investigated preceding the inhibition studies. DTT. The mixtures were incubated at 37 °C for 30 min. The incubations were terminated by addition of 200 µl ice-cold ACN. Sample preparation was performed as described above and the amount of pyrene 1-sulfate formed was determined using HPLC analysis (see above).

Reaction rates were calculated and plotted against the substrate concentrations to obtain Michaelis-Menten curves, and the 'one site binding hyperbola' fitting module of GraphPad Prism 3.0 was used to estimate K m and V max values.

SULT1E1 inhibition screening

A c c e p t e d M a n u s c r i p t 11 After optimizing the mSULT1E1 assay conditions, the ability of the assay to identify mSULT1E1 inhibitors was tested with a selection of 34 known EDCs. This selection included 19 EDCs which were used previously for hSULT1E1 inhibition screening under almost identical conditions [START_REF] Reinen | Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase[END_REF]. The reactions were initiated by adding 50 µl of 50 µM PAPS to 150 µl of a mixture containing 10 ng mSULT1E1, 250 nM OHP, and 2.5 µM of each library compound in a 100 mM phosphate buffer (pH 7.4) containing 2 mM EDTA and 1 mM DTT. The mixtures were incubated at 37 °C for 30 min and reactions were terminated by adding 200 µl ice-cold ACN. Sample preparation and determination of the amount of formed pyrene 1-sulfate by HPLC was performed as described above.

The 15 compounds in the EDC selection which had not been investigated for inhibition of the orthologous human enzyme, hSULT1E1, before were tested at the same concentration (2.5 µM) using the hSULT1E1 inhibition screening assay described previously [START_REF] Reinen | Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase[END_REF]).

IC 50 measurements

The IC 50 values were determined for a selection of 14 EDCs. These included the 10

EDCs for which IC 50 values had previously been determined for hSULT1E1 [START_REF] Reinen | Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase[END_REF] and four additional EDCs (testosterone, progesterone, resveratrol and enterolactone). In a total volume of 200 µl, 250 nM OHP, 50 ng/ml mSULT1E1, and 12.5 µM PAPS were incubated at 37 °C for 30 min in the presence and absence of various concentrations of the SULT1E1 inhibiting compounds. The incubations were terminated by addition of 200 µl ice-cold acetonitrile. Sample preparation and determination of the amount of formed pyrene-1-sulfate by HPLC was performed as described above. IC 50 measurements were also performed to A c c e p t e d M a n u s c r i p t enterolactone as described previously [START_REF] Reinen | Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase[END_REF]).

Computer modelling

The sequences of murine and human SULT1E1, PDB codes 1AQU [START_REF] Kakuta | mechanism. Crystal structure of a vanadate complex of estrogen sulfotransferase and mutational analysis[END_REF] and an E2-and PAP-bound hSULT1E1, were aligned and the structures superposed and compared, using Molecular Operating Environment (MOE). 1AQU is the murine structure cocrystallized with E2 and inactive cofactor PAP. Missing residues forming the loop Asp66 -Glu72 were modeled in using MOE. This loop is at the surface of the protein and far away from the active site. For comparison, automated docking calculations were performed for E2

and other ligands from this study to the substrate-free hSULT1E1 structure, using GOLD [START_REF] Jones | Development and validation of a genetic algorithm for flexible docking[END_REF]). The hSULT1E1 structure in complex with PAP and E2 [START_REF] Pedersen | Crystal structure of the human estrogen sulfotransferase-PAPS complex: evidence for catalytic role of Ser137 in the sulfuryl transfer reaction[END_REF] was kindly made available to us by Negishi et al. This structure is very similar to the PAPS-bound apo-structure of hSULT1E1, PDB code 1HY3 [START_REF] Pedersen | Crystal structure of the human estrogen sulfotransferase-PAPS complex: evidence for catalytic role of Ser137 in the sulfuryl transfer reaction[END_REF].

To further study the differences between the SULT1E1 structures, molecular dynamics simulations were performed on the resulting docked complexes using the GROMOS05 [START_REF] Christen | modulation of sulfotransferase 2A1 activity by celecoxib: product ratio switching of ethinylestradiol sulfation[END_REF] and GROMACS [START_REF] Berendsen | GROMACS: A messagepassing parallell molecular dynamics implementation[END_REF][START_REF] Lindahl | GROMACS 3.0: A package for molecular simulation and trajectory analysis[END_REF]) biomolecular simulation packages and the GROMOS force field parameter set 45A4 [START_REF] Schuler | An improved GROMOS96 force field for aliphatic hydrocarbons in the condensed phase[END_REF]. The cofactor was parameterized both in the active form, PAPS, and in the inactive form, PAP. Force field parameters are available in the Supplementary Material. Simulations were performed with both forms of the cofactor and the results were compared. After having established that the PAP-and PAPS-containing simulations displayed similar results, PAP was chosen as the preferred cofactor, to stay close to the crystal structure complexes. 10 simulations of 1 ns,

A c c e p t e d M a n u s c r i p t 13 starting from the same coordinates but using different random starting velocities, were performed for E2, OHP, E1, diethylstilbestrol (DES) and dienestrol (DIS) in complex with the PAP-bound dimeric form of hSULT1E1 as well as the PAP-bound mSULT1E1 monomer. To study the impact of dimerization of the human enzyme, 10 simulations were similarly performed for the monomeric form (chain A) of hSULT1E1 in complex with E2. To study the impact of crystal contacts in the hSULT1E1 structure, a 1 ns simulation of the periodic unit cell of the crystal structure, containing two dimers, was performed. All simulations were performed using the following protocol. Polar and aromatic hydrogen atoms were added to the protein-ligand complexes. The complexes were subsequently minimized and centred in a periodic box solvated with SPC water molecules [START_REF] Berendsen | Interaction models for water in relation to protein hydration[END_REF]. The minimal solute-towall distance was 0.9 nm. The systems contained approximately 13.000 molecules (monomer), 21.000 molecules (dimer) and 8.300 molecules (crystal). 2 (mSULT1E1), 7

(hSULT1E1, monomer) and 14 (hSULT1E1, dimer) counterions (Na + ) were added at random positions to obtain a system with a net charge of zero. After another round of energy minimization, initial velocities were randomly assigned according to a Maxwell-Boltzmann distribution at 50 K. The systems were heated up gradually, increasing the temperature by 50 K every 20 ps, followed by 40 ps of equilibration at 298 K. During the heating up of the system, position restraints with an initial force constant of 2.5 10 4 kJ mol -1 nm -2 on the heavy atoms were gradually reduced by a factor 10 every 20 ps. Subsequently, 1 ns of simulation was performed. For some of the systems extended simulations were carried out to further study their dynamic behaviour. A time-step of 2 fs was used and all bonds were constrained using the SHAKE algorithm [START_REF] Ryckaert | Numerical integration of cartesian equations of motion of a system with constraints: Molecular dynamics of n-alkanes[END_REF]. All simulations were conducted at constant temperature and pressure, using the weak coupling algorithm [START_REF] Berendsen | Molecular dynamics with coupling to an external bath[END_REF]. The solute and solvent molecules were separately coupled to two temperature baths at 298 K with a relaxation time of 0.1 ps. The relaxation time for the isotropic pressure scaling was set to 0.3

A c c e p t e d M a n u s c r i p t 14 ps with an isothermal compressibility of 2.807*10 -5 atm -1 and a reference pressure of 1 atm.

Non-bonded interactions were calculated using a triple range cutoff scheme. All interactions within 0.8 nm were calculated every time-step using a pair-list generated every fifth time-step.

Long-range interactions, up to 1.4 nm, were calculated every fifth time-step. A reaction-field term was added to the energies and forces, with an effective dielectric constant of 61.0 to represent the electrostatic interactions outside the 1.4 nm cut-off [START_REF] Tironi | A generalized reaction field method for molecular-dynamics simulations[END_REF]. Coordinates were stored every 0.4 ps for the solute. All trajectories were analyzed using the GROMOS05 [START_REF] Christen | modulation of sulfotransferase 2A1 activity by celecoxib: product ratio switching of ethinylestradiol sulfation[END_REF] and GROMACS [START_REF] Berendsen | GROMACS: A messagepassing parallell molecular dynamics implementation[END_REF][START_REF] Lindahl | GROMACS 3.0: A package for molecular simulation and trajectory analysis[END_REF]) biomolecular simulation packages.

A c c e p t e d M a n u s c r i p t

Validation of the mSULT1E1 inhibition assay

HPLC analysis confirmed the production of pyrene 1-sulfate from OHP in the presence of mSULT1E1, as described previously [START_REF] Reinen | Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase[END_REF]). The protein-and timedependent formation of pyrene 1-sulfate from OHP was investigated. The formation of pyrene 1-sulfate by mSULT1E1 is linear (R 2 = 0.99) with increasing protein concentration up to 94 ng/ml. The formation of pyrene 1-sulfate by mSULT1E1 at a protein concentration of 25 ng/ml was linear in time for at least 30 min (R 2 = 0.97). See Figure S1 in the supplementary materials. The sulfo-conjugation of OHP by mSULT1E1 displayed typical Michaelis-Menten kinetics (R 2 = 0.98), see Figure S2. Apparent K m and V max values measured by the HPLC assay were 305 ± 31 nM and 140 ± 6 pmol/min/µg mSULT1E1 (means ± SD), respectively.

No substrate inhibition could be observed within the tested range of substrate concentrations (up to 1 µM).

mSULT1E1 and hSULT1E1 inhibition screening and IC 50 measurements

In order to identify mSULT1E1 inhibitors, a selection of 34 known EDCs, see Figure 3, was screened at a 2.5 µM concentration using the optimized mSULT1E1 inhibition assay conditions. Compounds were considered to be strong inhibitors when more than 50% inhibition of mSULT1E1 was observed at an inhibitor concentration of 2.5 µM. 8 strong inhibitors were identified. E1, 17α-estradiol (αE2), E2, 17α-ethinylestradiol (EE2) and estriol (E3) were found to be very strong inhibitors displaying more than 95% inhibition. DES, 11-HMBP and 4-HEP were found to be strong inhibitors. DIS (72%), enterolactone (62%), zearalenone (58%), genistein (52%), resveratrol (46%), hexestrol (31%), 4-HEP (30%) and progesterone (25%) showed stronger inhibition for hSULT1E1 than for mSULT1E1. Testosterone and 6-HEBP displayed stronger inhibition for mSULT1E1 than for hSULT1E1, with differences of 38 and 29% respectively. For all the other compounds the inhibition difference between the species was less than 25%.

Based on the inhibition screening and species differences results, 14 out of the 34

EDCs, listed in Table 1, were selected for IC 50 measurements using the optimized mSULT1E1 inhibition assay. IC 50 values using the hSULT1E1 inhibition assay for testosterone, progesterone, resveratrol and enterolactone were measured as well. The results are displayed in Table 2. Using the IC 50 values, the K i values and the ratio between the mSULT1E1 K i and the hSULT1E1 K i values were calculated, using the Cheng-Prusoff equation. [START_REF] Cheng | Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction[END_REF]) From Table 2 it can be concluded that there are significant differences between the binding affinities of E1, DIS and enterolactone between mSULT1E1 and hSULT1E1, all of these compounds being weaker binders of mSULT1E1. A significant difference in binding affinity is here defined as a ratio of K i values significantly greater than 10. E1 and E2 both display similar binding affinities to mSULT1E1 as well as to hSULT1E1, which is in accordance with previous studies [START_REF] Falany | Enzymology of human cytosolic sulfotransferases[END_REF][START_REF] Hempel | Site-directed mutagenesis of the substrate-binding cleft of human estrogen sulfotransferase[END_REF]Song 2001). The difference in binding affinity of E1 originates from a slightly improved affinity for the human However, the changes in affinity between E2 and E1 in one protein are less than a power of ten. Interestingly, DIS, which is structurally very similar to DES, displays a similar binding affinity as DES to hSULT1E1, whereas DIS is a significantly weaker binder than DES in mSULT1E1. Enterolactone is a very weak binder of mSULT1E1, whereas it is a reasonably strong binder of hSULT1E1.

However, even for the significant differences in affinity, the differences are not large enough to realistically be able to rationalize them computationally. Therefore, the focus of the computational work remained on general structural and mechanistic differences between the murine and human enzymes.

Computer modelling

General structural observations

The sequence identity of mSULT1E1 and hSULT1E1 is 77% and overall the structures are very similar. A comparison reveals a conserved region, both in terms of sequence and conformation, near the cofactor and a more variable region on the opposite side of the binding site, near the surface of the protein. Figure 4 displays the active site of the human E2-bound structure superposed on the murine E2-bound structure (1AQU) [START_REF] Kakuta | mechanism. Crystal structure of a vanadate complex of estrogen sulfotransferase and mutational analysis[END_REF]. All amino acids in the active site that differ between the two proteins are displayed. A difference in the inner parts of the binding site is the substitution of Phe80 in hSULT1E1 for Tyr81 in mSULT1E1. This structural difference has been shown to be important for substrate specificity, by acting as a gate, preventing bulkier substrates, such as dehydroepiandrosterone [START_REF] Shevtsov | Crystallographic analysis of a hydroxylated polychlorinated biphenyl (OH-PCB) bound to the catalytic estrogen binding site of human estrogen sulfotransferase[END_REF]). In addition, mutagenesis studies have shown that Lys85 is not crucial for hSULT1E1 E2 sulfonation [START_REF] Hempel | Site-directed mutagenesis of the substrate-binding cleft of human estrogen sulfotransferase[END_REF]. Similar to hSULT1E1, mSULT1E1 also has two residues that form a salt bridge on this side of the binding site, but in a slightly shifted position; Arg23 and Asp21, corresponding to Asp22 and Tyr20 in hSULT1E1. In addition to these salt-bridge pairs, hSULT1E1 displays Lys10 located further away from the substrate The corresponding residue in mSULT1E1 is Val11. Another difference between the mSULT1E1 and the hSULT1E1 structures is in the loop containing residues 146-148 (hSULT1E1 numbering). The residues in this loop differ between hSULT1E1 and mSULT1E1, being more hydrophilic in the murine structure, see Figure 4 for details. Moreover, on position 142 (hSULT1E1 numbering) the Phe of hSULT1E1 is substituted for a Leu in mSULT1E1. In summary, hSULT1E1 has more aromatic and hydrophobic residues than mSULT1E1 in the buried parts of the binding site.

Molecular dynamics simulations of E2 in hSULT1E1 and mSULT1E1

Molecular dynamics simulations were initially performed for E2 bound to solvated mouse and human SULT1E1 in complex with PAPS, starting from the crystal structures.

Surprisingly, the substrate moved away from the catalytically active position at different times during the simulations. Different parameters, e.g. different ionization states, of the cofactor were tested, as well as performing the simulations with inactive cofactor PAP. In addition, Heparan sulfate N-sulfotransferase is not a cytosolic sulfotransferase and the catalytic histidine residue is substituted by a glutamate, most likely acting as a catalytic base.

Gorokhov et al did not observe a direct hydrogen bond between the substrate and the catalytic base during the simulation. Instead water-bridged hydrogen bonds were observed (Gorokhov 2000). Such movements were not observed in a previous QM/MM study on similar complexes, but these simulations included only very limited dynamics at maximally 200 K and with distance constraints between the substrate and the active site residues [START_REF] Lin | Searching for the minimum energy path in the sulfuryl transfer reaction catalyzed by human estrogen sulfotransferase: role of enzyme dynamics[END_REF]).

To investigate whether the observed effect was due to a simulation defect or a result of simulating the proteins free in solution as opposed to in a crystal, a crystal simulation was performed for hSULT1E1. Interestingly, the movement of the substrates in the crystal were significantly less than in the simulations of the proteins free in solution. Table 3 displays the percentage of time of the simulation in which the 3-hydroxyl oxygen of E2 is within 3.5 Å of His107Nε and the RMS deviation of the substrate atoms during the simulation. The corresponding values for the simulations of the human dimer in solution are also displayed in Table 3. The substrate is not forming a hydrogen bond with His107 during the simulation in any of the monomers in the crystal. However, the movement of the substrate is very small, with a total RMS deviation of only 1.2 Å, and this deviation is fairly constant over time, indicating that the substrate moves away only slightly from its initial position as observed in the crystal structure. In the dimer simulations the substrate displays more movement than in the crystal, as can be seen from the RMS deviation values. Note that the values for the dimer

Studies of the dimerization interface and the importance of the quaternary structure for substrate positioning and interactions in the active site

Most cytosolic sulfotransferases, with the exception of mSULT1E1, are homodimers.

The dimerization interface has been identified to be a short segment at the carboxyl terminus, containing the KXXXTVXXXE sequence. Mutation of the valine in this sequence to a glutamate has been shown to result in monomer formation and introducing this sequence in naturally occurring sulfotransferase monomers converts them to dimers (Petrotchenko 2001).

Although Zhang et al have argued that one of the active sites in the dimer functions as an allosteric site, which could be an explanation for substrate inhibition, several studies show that total activity and substrate inhibition is independent of subunit dimerization [START_REF] Zhang | ), S = substrate concentration. S(mSULT1E1) = 250 nM , S(hSULT1E1) = 18.75 nM. K m (mSULT1E1) = 305 nM, K m (hSULT1E1) = 6.4 nM. c Ratio is defined as K i[END_REF]. The same has also been observed for substrate affinity (Petrotchenko 2001;[START_REF] Pedersen | Crystal structure of the human estrogen sulfotransferase-PAPS complex: evidence for catalytic role of Ser137 in the sulfuryl transfer reaction[END_REF]Lu 2008;[START_REF] Lu | Identification and characterization of two amino acids critical for the substrate inhibition of human dehydroepiandrosterone sulfotransferase (SULT2A1)[END_REF]). Lu et al suggest that the main role for dimerization of sulfotransferases could be to maintain structural stability [START_REF] Lu | Identification and characterization of two amino acids critical for the substrate inhibition of human dehydroepiandrosterone sulfotransferase (SULT2A1)[END_REF]. The E2-containing hSULT1E1 structure crystallizes as a homodimer, but contains the V269E mutation which disrupts the dimer in solution. Simulations were made using this structure as well as one of the monomers of the hSULT1E1 and results of the ligand positioning and interactions were compared (Table 4). It can be seen that the dimer and monomer simulations behave very similarly in terms of substrate interaction with His107 throughout the simulations. The RMS deviation of the substrate is somewhat higher in the monomer simulations than in the dimer ones. These results indicate that the time the substrate stays in the catalytic position is similar in the monomer and the dimer simulations, but when the substrate moves, it moves slightly more in the monomer than in the dimer. Note that the results are average values over 10 simulations. No significant differences were observed between the RMS fluctuations of the backbone atoms in the simulations of the dimer and the monomer. Differences in the fluctuations of side-chain atoms of active site residues were observed for Tyr20 and Lys85, which displayed larger average fluctuations in the monomer simulations.

To study the stability of the human monomer in solution compared to the dimer, as well as the stability of the dimer in solution, the secondary structure was monitored during the simulations. The dimer displayed stable secondary structure during the simulations.

Differences between the monomer and the dimer simulations were observed for the helix consisting of residues 64-69 on the surface of the protein and for the helix consisting of residues 261-264, leading up to the dimer interface, see Figure 5. These helices appeared to be less stable in the monomer simulation.

These results in combination with the previously reported results for SULT1E1 and other sulfotransferases, where no difference between natural dimers or mutated monomers A c c e p t e d M a n u s c r i p t 22 could be found for substrate activity or substrate inhibition, leads us to believe that the substrate interactions and positioning in the active site is not a direct result of the quaternary structure of the protein.

The interactions between the monomers at the dimer interface were studied in more detail. In the wild-type hSULT1E1 structure, PDB code 1G3M [START_REF] Shevtsov | Crystallographic analysis of a hydroxylated polychlorinated biphenyl (OH-PCB) bound to the catalytic estrogen binding site of human estrogen sulfotransferase[END_REF], there are 6 hydrogen bonds between the monomers; Lys264O(A)-Ala270H(B), Phe267O(A)-Val269H(B), Lys264NZ(A)-Glu273OE2(B) and the inverse for the other monomer. In the simulations both the hydrogen bonds between Lys264 and Glu273 are completely disrupted, in accordance with the crystal structure of the V269E mutant, see Figure 5 for details.

Moreover, during the course of the simulations water molecules enter the interface and some of the direct hydrogen bonds are substituted with interactions with water, bridging the interactions between the monomers. The distance between the monomers was measured in simulations of each of the following ligands bound to hSULT1E1: E2, E1, OHP, DES and DIS. Figure 6 displays the distance between the monomers, calculated as the distance between the centres of geometry of the α-carbons of the backbone of residues 264-273 of the two monomers, averaged over 10 1 ns simulations. The distance increases from 6.5 Å initially up to 9.5 Å at the end of the simulations and is continuously increasing. The same distance in the crystal simulation stays around 6.5 Å throughout the simulation. Note that the simulation times are too short for any significant structural changes to occur, as confirmed by the conserved secondary structure throughout the dimer simulations. However, it seems that the initial stages of monomerization of the SULT1E1 V269E mutant can be observed. The hydrogen bonds between the other oxygen of the ligand and Asp22/Lys85 (hSULT1E1) and Arg23/Asn86 (mSULT1E1) on the opposite side of the binding pocket were also monitored and are displayed in Table 5. For DES and DIS in hSULT1E1 the hydrogen bond with His107 is substituted by hydrogen bonds with Asp22 and Lys85, whereas for the other ligands and for the murine protein simulations it is less clear if that is the case. In order to determine whether it would be possible for the ligands to form hydrogen bonds with the two oxygen/hydroxyl-groups and the protein simultaneously, the distances between A c c e p t e d M a n u s c r i p t 24 His107/108Nε on one side of the binding pocket and Asp21Cγ(human)/Arg22Cζ(murine) as well as Lys84Nζ(human)/Asn85Cγ(murine) were monitored during the simulations (table 5).

Differences between mSULT1E1 and hSULT1E1

The distances between the two oxygen atoms in the ligands (e.g. 3-OH and 17-OH for E2)

were also monitored. The shortest distance in mSULT1E1 is approximately 13.5 Å in all simulations, whereas the shortest distance in hSULT1E1 is 15.2 Å. The ligand O-O distance is similar in the mSULT1E1 and hSULT1E1 simulations. It can be expected that E2 and E1 will not form the two hydrogen bonds simultaneously in the human protein, whereas this could be possible in the murine protein. For DES and DIS that display a longer distance between the two oxygens, these hydrogen bonds could be formed simultaneously in both the human and murine proteins. The percentage of simulations in which ligand-protein hydrogen bonds at both sides of the binding pocket are observed simultaneously (table 5), is significantly higher in mSULT1E1 than in the human protein. As expected, in the human protein it is mostly for DES and DIS that both hydrogen bonds occur.

Experimentally, E2 displays similar affinity to hSULT1E1 and mSULT1E1. However, the substrate stays longer in the catalytic position in the murine protein. The trajectories of the simulations show that the substrate in the hSULT1E1 simulations moves away from the active site to a position where it can form hydrogen bonds with Asp22 and Lys85. This part of the pocket also offers a hydrophobic environment for the rest of the substrate, more so than in the murine protein. DES is a substrate for both human and murine SULT1E1, but displays a significantly lower activity in the human protein [START_REF] Falany | Enzymology of human cytosolic sulfotransferases[END_REF]Kakuta 1998a). However, the inhibition of both enzymes by DES is similar, as displayed in Table 2. The existence of a second binding site/binding pose within the active site of hSULT1E1 could explain this observation. The 10fold longer time that DES stays in the catalytic position in the murine protein and the high percentage of hydrogen bond formation with Asp22 and Lys85 in the human protein all point to the substrate moving away quickly from the catalytically active position in hSULT1E1 to a second, catalytically inactive, binding site displaying a similar or stronger binding affinity.

DIS is not a substrate for bovine SULT1E1, but has to our knowledge not been tested in murine or human SULT1E1. DIS and DES display similar affinity to the human protein, see Table 2, but the affinity of DIS in mSULT1E1 is 30 times weaker than that of DES. No direct reason for this could be suggested from the data in Table 5.

In summary, the ligands in the murine protein stay longer in the catalytically active pose during the simulations than in the human protein. This is most probably a result of the opportunity to form hydrogen bonds on both sides of the active site in mSULT1E1, which is not possible to the same extent in hSULT1E1. The ligands in hSULT1E1 seem to move to a sub-pocket, offering hydrophobic interactions as well as possibilities to form hydrogen bonds with Asp22 and Lys85. In mSULT1E1 the movement is more random after the substrate has left the catalytically active pose. It is possible that the human protein offers two distinct binding sites, whereas the murine protein only has one. This offers an explanation to the more favourable binding affinity for the human protein of the ligands tested in this study as well as a possible explanation to substrate inhibition observed in hSULT1E1, but not in mSULT1E1.

A c c e p t e d M a n u s c r i p t

The aim of this study was to investigate the differences in inhibition by EDCs of murine and human estrogen sulfotransferase in vitro and in silico and to study whether the observed differences could give suggestions as to reasons for differences in substrate inhibition and quaternary structure of these two proteins.

Human SULT1E1 displays substrate inhibition at higher concentrations of different substrates (Falany 1995;[START_REF] Reinen | Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase[END_REF], whereas no substrate inhibition has been observed for murine SULT1E1. (Kakuta 1998a) This was confirmed here for OHP, as no substrate inhibition was observed in murine SULT1E1 in the concentration range tested. This again indicates that the OHP-assay is very similar to an estradiol-based assay, with the significant difference that no radio-active compounds are required to perform the experiments [START_REF] Reinen | Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase[END_REF].

Based on the inhibition screening results of 34 EDCs and the previously determined IC 50 values for hSULT1E1, 14 EDCs were selected for IC 50 value measurements. The binding affinities (K i values) were similar for most of the compounds, except for E1, DIS and enterolactone, which displayed 20 to 50 times higher affinity for hSULT1E1. Large similarities between the two enzymes with regards to the bioactivation of a large set of SULTdependent promutagens have also been found previously (H.R. Glatt, unpublished results).

The selection of 34 EDCs that were screened includes five structurally related polycyclic compounds which were bioactivated to mutagens by hSULT1E1 and mSULT1E1 at up to 200-fold different levels (H.R. Glatt, unpublished results). Interestingly, only small differences were observed in affinity for human and murine SULT1E1 for these compounds. In the simulations of the natural substrate, E2, in complex with both PAPS-and PAPbound hSULT1E1 and mSULT1E1, the substrate moved away from the catalytically active position, observed in the crystal structures. Interestingly, the substrate movement in the simulation of a unit cell of the hSULT1E1 crystal structure was significantly less, indicating that the substrate movement in the proteins free in solution is not a simulation defect.

Differences in movement of the backbone in the free and crystal simulations were observed and included two regions close to the binding site, residues 1-20 and 215-230. Pedersen et al observed that crystal contacts in the structure of SULT2A3 could be the reason that the substrate, DHEA, was unable to bind to the protein, although the crystal was soaked with high concentrations of the substrate [START_REF] Pedersen | Crystal structure of SULT2A3, human hydroxysteroid sulfotransferase[END_REF]. They observed that considerable differences in the conformations of residues [18][19][20][21][22][23][82][83][84][85][86][87][88][89][90] exist between human SULT2A3 and mSULT1E1 and that e.g. the loop of the corresponding residues 239-252 in SULT2A3 would have to change conformation in order to accommodate DHEA in a similar orientation as E2 in mSULT1E1. A number of substrate/inhibitor bound sulfotransferase crystal structures are the result of crystal soaking experiments [START_REF] Kakuta | mechanism. Crystal structure of a vanadate complex of estrogen sulfotransferase and mutational analysis[END_REF]Kakuta 1998b;[START_REF] Pedersen | Crystal structure of SULT2A3, human hydroxysteroid sulfotransferase[END_REF][START_REF] Pedersen | Crystal structure of the human estrogen sulfotransferase-PAPS complex: evidence for catalytic role of Ser137 in the sulfuryl transfer reaction[END_REF][START_REF] Shevtsov | Crystallographic analysis of a hydroxylated polychlorinated biphenyl (OH-PCB) bound to the catalytic estrogen binding site of human estrogen sulfotransferase[END_REF]), which will likely not allow for large conformational changes to occur upon ligand binding. Interestingly, some substratebound crystal structures that are the result of co-crystallization experiments display either multiple ligands in the binding site or several possible orientations of the ligand [START_REF] Rehse | Crystal structure of human dehydroepiandrosterone sulphotransferase in complex with substrate[END_REF];

A c c e p t e d M a n u s c r i p t present simulations, this suggests that there are several favourable binding poses in sulfotransferases, possibly not observed in the ligand soaked crystal structures. Multiple binding orientations in the active site, one of them catalytically inactive, has also been suggested to explain substrate inhibition at higher substrate concentrations. When the inactive binding orientation becomes more occupied at higher substrate concentrations this may block the substrate from binding in the catalytically active pose.

Similarly to many other cytosolic sulfotransferases, hSULT1E1 has been shown to be biologically active as a dimer. In contrast, mSULT1E1 is active as a monomer. Zhang et al [START_REF] Zhang | ), S = substrate concentration. S(mSULT1E1) = 250 nM , S(hSULT1E1) = 18.75 nM. K m (mSULT1E1) = 305 nM, K m (hSULT1E1) = 6.4 nM. c Ratio is defined as K i[END_REF] proposed that one of the binding sites in the hSULT1E1 dimer is catalytic and the other site is allosteric and regulates turnover. However, it has also been shown that activity, affinity and substrate inhibition are independent of subunit dimerization (Petrotchenko 2001;[START_REF] Pedersen | Crystal structure of the human estrogen sulfotransferase-PAPS complex: evidence for catalytic role of Ser137 in the sulfuryl transfer reaction[END_REF]Lu 2008;[START_REF] Lu | Identification and characterization of two amino acids critical for the substrate inhibition of human dehydroepiandrosterone sulfotransferase (SULT2A1)[END_REF]. It has been suggested that the role of dimerization is to maintain structural stability [START_REF] Lu | Identification and characterization of two amino acids critical for the substrate inhibition of human dehydroepiandrosterone sulfotransferase (SULT2A1)[END_REF]. The simulations performed in this study show no differences in ligand (E2) movement or interactions in the hSULT1E1 monomer and dimer. The secondary structure is maintained throughout the simulations of dimeric hSULT1E1. In contrast, the human monomer simulations display disruption of the secondary structure in helices on the surface and leading up to the dimer interface, confirming that dimerization could have an effect on structural stability.

The differences in ligand-protein interactions and ligand movements during the simulations of ligand-bound hSULT1E1 and mSULT1E1 were also studied. In general, the ligands stayed longer in their catalytically active position in mSULT1E1 than in hSULT1E1.

Based on distance and hydrogen bond analysis we suggest that this observation is explained A c c e p t e d M a n u s c r i p t 29 by the ability of the ligands to form hydrogen bonds with both its hydroxyl-groups simultaneously in mSULT1E1, whereas this is not possible to the same extent in hSULT1E1.

The movement of the ligands, when having moved away from the catalytically active position, is larger in the murine than in the human enzyme. Analysis of the trajectories of the simulations suggests that the ligands in the human protein move to a sub-pocket in the active site allowing for interaction with Asp22 and Lys85 as well as offering hydrophobic interactions. The movement of the ligands in the mSULT1E1 active site is more random.

Lys85 has been shown not to be crucial for activity in hSULT1E1 [START_REF] Hempel | Site-directed mutagenesis of the substrate-binding cleft of human estrogen sulfotransferase[END_REF]. The K m for E2, E1 and DHEA even decreased slightly in the K85A mutant compared to the wild-type.

Our in silico observations agree with these studies, as we are proposing a second non-catalytic binding pose in the active site possibly made less favourable due to the mutation. A similar effect was observed for the corresponding mSULT1E1 mutant, N86A [START_REF] Petrotchenko | Substrate gating confers steroid specificity to estrogen sulfotransferase[END_REF].

As the activities of E2 in hSULT1E1 and mSULT1E1 are similar, we do not suggest that the ability of this ligand to form hydrogen bonds on both sides of the binding pocket simultaneously is affecting activity, but merely the affinity of the ligands in the catalytically active position. Additional favourable binding orientations for some ligands in hSULT1E1

would offer an explanation of why DES shows significantly lower activity in hSULT1E1 than in mSULT1E1, but displays similar binding affinities to the two proteins. Our simulations suggest that DES moves away very quickly from the catalytically active pose in hSULT1E1, whereas it stays significantly longer in mSULT1E1. A favourable second binding pose in hSULT1E1 would explain the similar binding affinities in hSULT1E1 and mSULT1E1. The substrate OHP, which has only one hydrogen bonding moiety, is the only compound that behaves similarly in the two proteins. However, the activity for this substrate is almost 50 times lower in mSULT1E1 compared to hSULT1E1. M a n u s c r i p t A c c e p t e d M a n u s c r i p t

Step 1

Step 2

Step 3

  the mechanism of SULT1E1, as well as to identify EDCs that inhibit this enzyme.

  Germany). Acros (Geel, Belgium) supplied nonylphenol. HPLC-grade acetonitrile (ACN), 3'phosphoadenosine-5'-phosphosulfate (PAPS), 1-hydroxypyrene (OHP), all inhibitors and all other chemicals were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands).The syntheses of (±)-4-(α-hydroxyethyl)pyrene (1-HEP), 6-hydroxymethylbenzo[a]pyrene (6-HMBP), 11-hydroxymethylbenzo[a]pyrene (11-HMBP), (±)-6-(αhydroxyethyl)benzo[a]pyrene (6-HEBP) and 10-hydroxycyclopenta[mno]benzo[a]pyrene (10-HCPBP) will be described elsewhere (C. Donath et al., to be published). All these benzylic alcohols were bioactivated to mutagens by hSULTs and mSULTs (C. Donath et al., to be published) and therefore are expected to compete with the conjugation of other substrates by these enzymes.

  the library of tested EDCs had not been screened previously in the hSULT1E1 inhibition assay.[START_REF] Reinen | Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase[END_REF] These compounds were screened for hSULT1E1 inhibition and 4 strong inhibitors were identified; 4-HEP, daidzein, resveratrol and enterolactone, as displayed in Figure3.

  slightly decreased affinity for the murine protein when compared to estradiol.

  17β-OH of E2 can form a hydrogen bond with Asn86 in mSULT1E1. In hSULT1E1 this position is occupied by Lys85, which in turn forms a salt bridge with Asp22, as displayed in Figure4. Lys85 is pointing away from the binding site in the human apo-structure, but it displays significant flexibility both in terms of B-factors in crystal structures and during molecular dynamics simulations

  to up to 4 ns. However, the substrate still moved away from the catalytically active position observed in the crystal structures. A similar observation was made by Gorokhov et al when simulating heparan sulfateN-sulfotransferase (Gorokhov 2000).

  10 simulations, whereas the crystal simulation was only performed once, and the values are an average over the 2 identical monomers in the simulated unit cell.To study which parts of the protein were more flexible in the simulation of the dimer free in solution as compared to the crystal, the RMS fluctuations of the backbone of the protein were monitored. The parts of the protein that fluctuate more in the simulation of the free dimer are residues 1-20, 60-70, 110-120 and 215-230. The residues near the N-terminal as well as residues 215-230 could have direct influence on the shape and interactions of part of the active site.

  experimental inhibition data, 5 compounds were simulated in complex with both murine and human SULT1E1; E2, E1, DES, DIS and the substrate used in the enzyme assay, OHP. All simulations were performed 10 times for 1 ns in each protein. The ligands were first positioned in a position compatible with catalytic activity. However, all five ligands moved away from this position in a number of simulations. Table5 displaysthe average occurrence of a hydrogen bond between the hydroxyl-group of the ligand and His107Nε as well as the average RMS deviation of the ligand atoms. With the exception of OHP, the compounds stay on average longer in the catalytic pose in the murine protein. To study whether the movements of the ligands are similar in the murine and human proteins, the average fluctuations of the atoms of E2 were calculated. As displayed in Figure 7, the fluctuation pattern is similar for all simulation conditions with the exception of mSULT1E1. E2 displays more equal fluctuations throughout the molecule in the murine protein, whereas the ligand atom fluctuations in the human protein show a minimum in the middle of the molecule, indicating a "twisting" movement around the centre of the molecule. The average fluctuations are smaller in the murine protein, suggesting that it stays in a more stable position for a longer period of time than the ligand in the human protein. Table 5 also shows the percentage of the simulations where the catalytically important hydrogen bond is present for more than 50% of the simulation.

  Figure 8 displays the last snapshots of 10 simulations of E2 in hSULT1E1 and mSULT1E1. More substrates are still located in the catalytically active position in the murine protein simulations, whereas those substrates that have left this position are fairly spread over the binding pocket. In contrast, fewer substrates are located in the catalytically active position in hSULT1E1. Moreover, the substrates in a different position than the catalytically active ones seem to gather in one sub-pocket of the active site, offering hydrophobic interactions as A c c e p t e d M a n u s c r i p t 25 well as hydrogen bond opportunities with Asp22 and Lys85. Similar behaviour was observed for the other ligands simulated in this study.

  murine and human SULT1E1 structures suggests that their binding sites are very similar. Differences involve differences in hydrogen bond-forming residues in the outer part of the binding site as well as a larger amount of hydrophobic and aromatic residues in the buried part of the binding site of the human SULT1E1 structure.

  shown to be a strong inhibitor of sulfotransferases, strengthening the hypothesis of substrate inhibition due to the formation of dead-end complexes[START_REF] Rens-Domiano | Inhibition of M and P phenol sulfotransferase by analogues of 3'-phosphoadenosine-5'-phosphosulfate[END_REF][START_REF] Zhang | ), S = substrate concentration. S(mSULT1E1) = 250 nM , S(hSULT1E1) = 18.75 nM. K m (mSULT1E1) = 305 nM, K m (hSULT1E1) = 6.4 nM. c Ratio is defined as K i[END_REF]. It is therefore surprising that mSULT1E1 does not seem to display substrate inhibition. However, murine SULT1E1 has been studied significantly less than hSULT1E1. It is possible that substrate inhibition and non-competitive inhibition is a combination of effects, both dead-end complex formation and a second favourable, but nonproductive, binding orientation in the active site. More studies, e.g. mutations of the 85/86 and 22/23 positions in the hSULT1E1 and mSULT1E1 proteins and studies of substrate inhibition patterns in the mutants could shed more light on this issue.

Figure 1 .

 1 Figure 1. The reaction mechanism of sulfotransferases is proposed to be a sequential

Figure 2 .

 2 Figure 2. His107 accepts a proton form the hydroxyl group of the substrate, which can

Figure 3 .

 3 Figure 3. Inhibition screening of 34 compounds with known estrogenic properties.

Figure 4 .

 4 Figure 4. Differences between mSULT1E1 (pink) and hSULT1E1 (grey). Both structures

Figure 5 .

 5 Figure 5. (A) The human SULT1E1 dimer. PAP is displayed in ball and stick and E2 in

Figure 6 .

 6 Figure 6. Distance between the centres of geometry of α-carbons of the backbone of residues

Figure 7 .

 7 Figure 7. RMS fluctuation of the atoms of E2 simulated in complex with human SULT1E1,

  over all atoms are 1.03, 1.13, 1.22 and 0.98 Å for hSULT1E1 A, B, hSULT1E1 monomer and mSULT1E1, respectively.

Figure 8 .

 8 Figure 8. The last snapshots of 10 1 ns simulations of E2 in human (A) and murine (B)

  Percentage of time that the 3-hydroxyl of E2 is within 3.5 Å of the Nε of the catalytic histidine, His107, and the RMS deviation of the substrate atoms during simulations of the human dimer and the human crystal.

  Figure 1A

  Figure 2

  Figure 5A

  Figure 5B

Table 1 .

 1 Structures of 14 EDCs selected for IC50 measurements.

	1

Table 4 .

 4 Average occurrence of a hydrogen bond between the 3-hydroxyl of E2 and His107Nε, and the RMS deviation of the substrate atoms during simulations of the SULT1E1 human dimer and the free human monomer A.

		Human SULT1E1 dimer	Human SULT1E1 crystal
		Monomer A 1	monomer B 1	monomer A 2	monomer B 2
	% time 3β-OH < 3.5 Å from His107Nε	38.1	58.7	10.8	60.8
	RMS deviation substrate (Å)	2.1	1.8	1.2	1.2
	1 Average over 10 simulations
	2 Average over two identical monomers in the crystal

Acknowledgements

The authors thank Prof. Negishi for making the 17β-estradiol/PAP SULT1E1 complex structure available.

Supplementary material

b Percentage of independent simulations in which the hydrogen bond to His107Nε (human) or His108Nε (mouse) occurs for more than 50% of the simulation time.

c Average occurrence of a hydrogen bond between the ligands and Lys84Nζ (human) or Asn86Nδ (mouse).

d Average occurrence of a hydrogen bond between the ligands and Asp22Oδ (human) or Arg23N/ (mouse).

e Average distance between Lys84Nζ and His107Nε (human) or Asn86Nδ and His108Nε (mouse). g Average distance between the two hydroxyl O-atoms in the ligands.

h Percentage of independent simulations in which both hydroxyl groups of the ligands are simultaneously engaged in hydrogen bonds with the protein.

i Average RMS deviation of the ligand over the simulations. The average is taken over 10 simulations for each ligand.

A c c e p t e d M a n u s c r i p t