Eur. J. Biochem. 88, 287-295 (1978)

Epiarginasic Regulation in Saccharomyces cerevisiae Citrulline, the Third Effector, Acts at a Specific Binding Site on the Ornithine Carbamoyltransferase

Jean-Paul SIMON and Victor STALON

Laboratoire de Microbiologie, Faculte des Sciences, Universite Libre de Bruxelles, and Institut de Recherches du Centre d'Enseignement et de Recherches des Industries Alimentaires et Chimiques, Bruxelles

(Received January 9, 1978)

The general purpose of this paper is to define the molecular process of the epiarginasic inhibition of ornithine carbamoyltransferase in Saccharomyces cerevisiae. We present here evidence for the existence of three isosteric sites for ornithine on the transferase. We have shown previously that one site is responsible for the catalytic power. The second site has been shown to be the regulatory site, which is actually implicated in the inhibition of catalysis when the enzyme is associated to epiarginase. A third site, the binding site, the subject of this paper, induces the necessary conformational transition in order to permit the association of the transferase with the epiarginase. This site can be saturated by ornithine or by citrulline. In the first case it induces the building up of a bimolecular complex made up of one molecule of ornithine carbamoyltransferase associated with one molecule of arginase. In the second situation the site, occupied by citrulline is responsible for the formation of a trimolecular complex in which one molecule of arginase is associated with two molecules of transferase.

This is supported by molecular sieving analysis on Sephadex G-200 SF, by inhibition tests of the ornithine carbamoyltransferase regulatory analysis and by the molecular composition of the several inultienzyme complexes induced by the effectors arginine, citrulline and ornithine.

Former studies describing the epiarginasic regula- tion of Saccharomyces cerevisiae ornithine carbamoyl- transferase [1,2] have shown that inhibition of the transferase activity in the presence of arginase is in- duced by the association of the two enzymes. This perfectly reversible association is under the control of two specific metabolic effectors which are arginine and ornithine [3].

Ornithine acts at a regulatory site on the ornithine carbamoyltransferase. Evidence of this site was given by specific acetylation of the enzyme [2]. It seems that the regulatory site of ornithine is responsible for sub- strate inhibition of the transferase as well as for its conformational transition bringing the trimeric en- zyme [4] into a structure which can recognize the arginase [2].

Arginine acts also at an allosteric site on arginase [5 ] . The saturation of this regulatory site by arginine occurs without effect on the catalytic power of arginase,

Enzymes. Ornithine carbamoyltransferase (EC 2.1.3.3); argin- ase (EC 3.5.3.1); urease (EC 3.5.1.5); catalase (EC 1.11.1.6); hexo- kinase (EC 2.7.1.1); alkaline phosphatase (EC 3.1.3.1).

but it reinforces the inhibition of the transferase by arginase [6].

MATERIALS AND METHODS

Enzymes

Ornithine carbamoyltransferase from S. cerevisiae was used in its penultimate stage of purification as previously described [4]. The specific activity of this enzyme preparation was of 24 200 units/mg protein (1 unit of ornithine carbamoyltransferase activity is defined at 30 "C as the amount of enzyme which cata- lyses the formation of 1 kmol citrulline/h).

Activity determinations were carried out with Tris/maleate 50 mM at pH 8.0, 10 mM ornithine and 1 mM carbamoylphosphate to yield a final volume of 2 ml. The reaction was started by addition of carba- moylphosphate after preincubation of the reaction mixture for 3 min at 30 "C. The reaction was terminat- ed by addition of 2 ml of 1 M HC1 and the total reac- tion mixture was taken for the colorimetric citrulline analysis [7]. Below the citrulline range of 0.1 pM,

288 Epiarginasic Regulation in S . cerevisiar

not detectable by the colorimetric assay, or in the presence of arginase, arginine and urea, the radio- metric assay was used as previously described [8]. In these conditions the amounts of citrulline measured are proportionally linear with the time of incubation and the amount of enzyme used.

The preparation of S. cerevisiae arginase used in this work is a highly purified preparation obtained by slight modifications of the procedure previously de- scribed [4]. These modifications consist in the general use during all the procedure of 1 mM MnCl2, a shorter (2 min) and more acidic (pH 6.5) thermodena- turation at 60 "C, an ammonium sulfate precipitation fraction between 30 and 55% saturation and the storage of the pure enzyme in maleate/NaOH buffer 1 mM at pH 6.5, 1 mM MnC12 and 400 mM KCI at - 20 "C. Under these conditions the yield of purifica- tion is 82% with a specific activity of 41 170 units and the preparation free of arginine remains stable for several months. Arginase activity is expressed as pmol urea synthesized/h in the experimental condi- tions previously described [4].

Ornithine carbamoyltransferase from E. coli has been partially purified following the five first steps of the procedure described by Legrain and Stalon [8]. The catabolic transferase from Pseudomonas putida [9], Bacillus lichenformis [lo] and Streptococcus faecalis [lo, 111 were also pure preparations. The anabolic enzymes from B. subtilis and B. licheniformis were crude extract preparations.

Arginase from B. lichenijormis was purified as previously described [12] and the enzyme from rat liver was purified following the procedure described by Schimke [I 31.

Inhibition Tests of the Ornithine Carbarnojiltransferases

The general procedure for carrying out the inhi- bition tests is quite similar to that proposed previously [2,3,6], as all the tests employ radiometric assay, there is no use of urease treatment and trichloroacetic precipitation. All the tests were done at pH 8.0 and 30 "C in Tris/maleate buffer, 50 mM, except for the catabolic transferase from P. putida which was tested at pH 7.5 in 20 mM potassium phosphate buffer [14].

Molecular Sieving Analysis on Sephadex G-200 SF

All the experiments were developed at 30 "C in 20 mM Tris/maleate buffer, pH 8.0, with 100 mM KCI. 0.9 x 30-cm jacketed chromatography columns were gently packed with swollen Sephadex G-200 SF. To assure a constant bed height and equilibration with eluant, the columns prior to each experiment were washed for 24 h with three times their total volumes

of equilibrium buffer containing the effectors at appro- priate concentrations. The exclusion volume is deter- mined from the elution volume ( VO) of a preparation of blue dextran. The effluent volume of the protein peak is taken as elution volume ( V,). Values of appa- rent molecular weight were assigned, based on cali- bration with : urease (480000), catalase (240000), alkaline phosphatase (78 000) and hexokinase (48 000), assuming the linear relationship between Ve/ VO and log M , as defined by Andrews [15].

For given conditions, the average day-to-day variations of VO was about ? 1% and a similar variation in V , might be expected. Analytical errors would produce a variation in molecular weight of

0.2 ml was applied on to the columns. The samples pre-equilibrated for 0.5 h at 30 "C contained all the effectors at the desired concentrations and the en- zymes. In all the experiments ornithine carbamoyl- transferase and arginase were taken in the ratio of 1 : 100 to maintain an excess of arginase. The total re- solution of the chromatogram took about 6 h in all cases, by using pumping systems which stabilize the elution speed at 12 ml/h.

& 3 % .

Determination of the Association Constants qf the Complexes between Arginase and Ornithine Carbamoyltransferase of S . cerevisiae on Sephadex G-200 SF

The concentration of arginase remains constant throughout this study and the ornithine carbamoyl- transferase concentration is varied.

0.150 ml of the sample was prepared 30 min before 0.1 ml was loaded on the columns. These experiments were conducted in 0.9 x 10-cm columns maintained at 30 "C. The elution time was 50 min. The fractions were directly collected drop by drop in the fraction tubes containing 1 ml reaction mixture made of 300 mM Tris/maleate buffer, pH 8.0, and 20 mM ornithine. Ornithine carbamoyltransferase activity was determined by addition of 2 mM carbamoyl- phosphate. Citrulline formed was detected either by colorimetric or radiometric assay.

The bound and unbound populations of ornithine carbamoyltransferase were estimated graphically by integrating the surface of the elution peak of the chromatogram.

Chemicals

The following chemicals are of purity grade and furnished by Sigma Chemical Co. (St Louis, Mo., U.S.A.) : arginine, ornithine, citrulline, Tris, maleic acid and carbamoylphosphate dilithium salt. Commer- cial citrulline was purified to remove ornithine im-

J.-P. Simon and V. Stalon 289

purity by the method described by Rivard and Carter [16]. Labeled ['4C]carbamoylphosphate was purchas- ed from New England Nuclear (Dreieichenhaini Frankfurt a. Main, F.R.G.). Potassium phosphate and KCI were from Merck, AG (Darmstadt, F.R.G.). Columns and gels used in the molecular sieving ana- lysis were furnished by Pharmacia Inc. (Uppsala, Sweden).

RESULTS

Citrulline, an Ejjector of the Arginase . Ornithine-Carbamoyltransferase Association

The association of ornithine carbamoyltransferase and arginase has been previously shown by molecular sieving. This association requires ornithine and ar- ginine as effectors [3] . When citrulline is used as effector instead of ornithine, the carbamoyltransferase activity is shifted to higher molecular weights (Fig. 1). The shift of carbamoyltransferase follows a titration curve for citrulline. The molecular weight of the free enzyme, with or without the effectors ornithine, citrulline or arginine, remains unmodified. Shift of the ornithine-carbamoyltransferase . arginase com- plex occurs only when arginine is present with one of the two other effectors, either ornithine or citrulline. The complex formed between the two enzymes in the presence of the effectors arginine and ornithine has an M , equal to 210000 2 20000. In the presence of arginine and citrulline the complex shifts to a molecular weight of 390000 (Table 1). The discre- pancy between the molecular weights of the two kinds of complexes implies different stoichiometries in the building up of the arginase and ornithine carbamoyl- transferase complex.

Effect of Citrulline on the Saturation of Ornithine Carbamoyltransferase by Arginase

As shown in Fig.2 it is clear that inhibition of the carbamoyltransferase by arginase is strongest in the presence of arginine and citrulline (curve c in Fig. 2).

Application of the model proposed earlier by Pen- ninckx and Wianie 161 gives a dissociation constant of 4.2 x lo-'' M for the complex induced by the effectors arginine and ornithine (Fig. 3A). In the pres- ence of citrulline and arginine, the curve is biphasic (Fig. 3 B). Two dissociation constants were measured, one for each branch of the biphasic plot obtained: they are 7.2 x lo-" M and 4.4 x lo-'' M. This latter observation leads to the conclusion that more than one molecule of ornithine carbamoyltransferase can bind to one molecule of arginase.

The Target of Citrulline Action

Does citrulline act on ornithine carbamoyltrans- ferase, on arginase or on both? It is well known that citrulline is competitive with respect to arginine at the arginine catalytic site [5]. The same is true with respect to ornithine at the catalytic site of ornithine carbamoyltransferase [I]. The regulatory arginine site on arginase has a much higher affinity for arginine than the catalytic site. Some analogues of arginine can identify both sites and some can only recognize the regulatory site or the catalytic site. Citrulline ap- pears to fall into the third category. Citrulline, or citrulline and ornithine promote neither the bind- ing of ornithine carbamoyltransferase and arginase (Table 1) nor the inhibition of ornithine carbamoyl- transferase (Fig. 2).

Arginase must be in its 'regulatory conformation' promoted by arginine in order to bind ornithine car- bamoyltransferase in the presence of ornithine or citrulline. In consequence the target of citrulline action is ornithine carbamoyltransferase.

Dissociation Constant o j the Complexes Induced by the Two Dgferent Pairs of Effectors, ArginineiOrnithine and ArgininelCitrulline

The dissociation constant of the ornithine-carba- moyltransferase . arginase complex in the presence of arginine and ornithine was estimated at 4.2 x lo-'' M. As this complex appears quite stable, it is possible to use molecular sieving to measure the dissociation constant of the associating system between arginase and ornithine carbamoyltransferase induced either by the effectors arginine and ornithine or by the effectors arginine and citrulline.

The integration of the data allows the making up of Scatchard plots [17] (Fig.4). Arginine (1 mM) and ornithine (20 mM), used as effectors, give a plot which shows that the ornithine-carbamoyltransferase . ar- ginase complex is a one-to-one association (15.5 pmol ornithine carbamoyltransferase are bound by 3 4.5 pmol arginase). The dissociation constant is 4 x M. In the presence of arginine (1 mM) and citrulline (20 mM) two kinds of complexcs could be detected between arginase and ornithine carbamoyl- transferase, as shown by the biphasic shape of the Scatchard plot (Fig.4B). The two slopes mean that there are two successive equilibria between arginase and carbamoyltranferase.

a) At low concentrations of carbamoyltransferase with regard to the concentration of arginase, the first equilibrium is a monomolecular association between the transferase and the arginase (7.3 pmol ornithine carbamoyltransferase bind to 7.3 pmol arginase) with a dissociation constant (Kd) of 3.8 x lop9 M.

b) When the majority of the arginase molecules are associated with one molecule of transferase, thus

290 Epiarginasic Regulation in S. cerevisiae

B E

c

Fig. 1. Effect of citrulline on the binding between ornithine carbamoyItransfi.ruse and arginase from Saccharornyces cerevisiae anaiy:ed by molecular sieving on Sephadex G-200. Chromatograms are expressed in units/ml as a function of the ratio Vc/Vo. (.---.) Omithine carbamoyltransferase activity; ( b m ) lo-' xarginase activity. Citrulline concentrations are: (A) 0 mM; (B) 0.5 m M ; (C) 2 m M : (D) 5 m M ; (E) 20 mM and (F) 50 mM. Arginine concentration was maintained constant in these experiments at 1.0 mM

J.-P. Simon and V. Stalon 291

Table 1. Action of the effectors arginine, ornithine and citrulline on the apparent molecular weight of ornithine carbamoyliransferase and arginase measured by molecular sieving on Sephadex G-200 SF columns (1.6 x 30 em) At pH 8.0 (see Materials and Methods) arginase and ornithine carbamoyltransferase concentrations are maintained constant throughout this work, at 250 units and 25 units, respectively

~

No Effectors x h.fr of arginase 10-3 x M , ~- of ornithine carbamoyltransferase

-~~~ arginine citrulline ornithine I I1 111 -

I I1 111

mM

135 135 135

20 135 135

20 135 20 135 20 135

-

-

-

-

125 125 125 125 -

- 125 -

I log [ Arginase] / M

Fig. 2. Action o j arginase on otnithine carbamoyftransferase ,from Saccharomyces cerevisiae. 0.9 pmol ornithine carbamoyltransferase was tested in the presence of 0.07-70 pmol arginase. The reaction mixture contained 50 mM Tris/maleate buffer, pH 8.0, 10 mM carbamoyl- phosphate and 20 mM ornithine. (A) Without effector or with 20 mM citrulline; (b) with 1 mM arginine; (c) with 1 mM arginine and 20 mM citrulline

for a concentration range of transferase equal or large- ly superior to the concentration of arginase employed, the second equilibrium describes the addition of a second transferase molecule to the preformed trans- ferase . arginase monomolecular couple (14.6 pmol ornithine carbamoyltransferase bind to 7.3 pmol ar- ginase). The dissociation constant of this step is of 1.8 x M and rules out the formation of a tri- molecular complex composed of 1 molecule of ar- ginase bound to 2 molecules of transferase (OCT).

Thus the building up of the ternary complex in the presence of citrulline follows a sequential mecha- nism of addition as described below :

OCT + arginase e (OCT) . arginase

(OCT) . arginase + OCT $ (0CT)z-arginase

IG = 3.8 x 10-9 M

K2 = 1.8 x M .

By using this sequential mechanism of formation of the complexes one can establish the theoretical Scatchard plot which delineates the phenomenon, described the dissociation constants K1 and K2 ex- perimentally estimated. In Fig. 4B this plot corres- ponds to the solid line and the experimental data are given by dots.

Desensitization

Chemical modification by acetylation of ornithine carbamoyltransferase has previously shown that the regulatory site for ornithine and the catalytic site are distinct [2]. It was supposed that this regulatory site played two distinct functions, to induce a conforma- tional change of the ornithine carbamoyltransferase, allowing its association with arginase, and simulta- neously to inhibit the catalytic power of ornithine

292 Epiarginasic Regulation in S. cerevisiae

A

O b 100 200 [Free arginase]- ’(nM-’)

B

8 c

O b 1 2 3 4 5 [Free arginasel- ’ (nM- ’ )

Fig. 3. Double-reciprocal plot oj titration data from Fig.2 plotted following the methods of Penninckx and Wiame [ 6 / . (A) With 1 mM arginine and 20 mM ornithine; (B) with 1 mM arginine, 20 mM ornithine and 20 mM citrulline

A

I

1 5 (Bound]/ [ f r e e ]

[ B o u n d ] / [ f r e e ]

Fig.4. Determination of’ the dissociation constant of the complex ornithine-carbarnoyltransferase arginase from Saccharomyces cere- visiae by molecular sieving on Sephadex G-ZOO. (A) Scatchard plot obtained when the effectors are 1 mM arginine and 20 mM orni- thine; arginase = 14.5 pmol; K d = 7 x lo9 M. (B) Scatchard plot obtained when the effectors are 1 mM arginine and 20 mM citrulline; arginase = 7.3 pmol; K1 = 4 x M; Kz = 1.8 x lo-’ M. For (A) and (B): pH 8; 30 “ C ; 100 mM KCI. Straight line is the calculated plot for the dissociation constants K1 and KZ experimen- tally determined; (0) the experimental data. [Bound]/[free] gives the ratio for ornithine carbamoyltransferase

Fig 5. Molecular sieving on Sephudi,.\. G-200 SF of’ uninhihitnbl~~ ornithine curbamo~l~ransf~ra.~e by arginase. (A) In the presence of 10 mM arginine and 20 mM citrulline. (B) In the presence of 1 mM arginine and 20 mM ornithine

3.-P. Simon and V. Stalon 293

W

c M m

.-

.-

- .- .. P i + a

s C M .-

2

\ 1 i- + + + + I \ I

I I I I I I I I I

i 2 a 3 M r - 0 0 0 0 " 7

v v m v r - z I v v

j I h ? 0 0 0 ? " ?

0 0 ~ ; ; O O "" x l v v v v v v v

v l m m m m m m w m

0 0 0 0 0 0 0 0 0 0 0

wlm wl m w l w l w l o m n m m m m m m N - , - - - e * . - - m -

0 0 8 8 8 8 8 8 8

cow m m r n m m r o m

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 w l o o m w l w l :z z z z z 2 2 2

carbamoyltransferase. However, it was found that a preparation of ornithine carbamoyltransferase from S. cerevisiae, kept six months at 4 "C in Tris/maleate, 50 mM, buffered at pH 8.0, loses 99% of its ability to be inhibited by arginase.

This modified ornithine carbamoyltransferase is re- cognized by arginase and either the bimolecular com- plex (in the presence of arginine and ornithine) or the trimolecular complex (when citrulline is substituted to ornithine; Fig.5) are formed. It is clear that (a) the binding properties of ornithine carbamoyltrans- ferase, (b) the regulatory properties (i.e. its ability to be inhibited by its association with arginase) and (c) the catalytic power are distinct properties which seem to be under the control of specific sites.

The Selectivity of Arginase Action

The binding of arginase to ornithine carbamoyl- transferase which has lost its regulatory properties opens the question of the selectivity of epiarginase in identifying carbamoyltransferase from various sour- ces. It has been clearly demonstrated that the arginase of S. cerevisiae inhibits the transferases of other yeasts which also possess this regulation [18]. Knowing that arginase and ornithine carbamoyltransferase are both trimeric, does the analogous quaternary structure play a drastic role in the 'identification' of the two enzymes?

With a view to answering this question, we tested the inhibition of several transcarbamoylases by the arginase of S. cerevisiae. We used catabolic ornithine carbamoyltransferases from Pseudomonas putida, Streptococcus faecalis and Bacillus licheniformis, which are respectively octameric [9], hexameric [lo, 111 and trimeric [lo]. We also tested anabolic enzymes which are trimeric such as these of E. coli W [81, B. licheni- ,formis and B. subtilis [lo, 181.

Only trimeric carbamoyltransferases can be in- hibited by arginase from s. cerevisiae and only in the presence of arginine (Table 2). It seems that the trimeric structure of the carbamoyltransferase is important in the 'identification' of the enzyme by the trimeric arginase. Between these trimeric ornithine carbamoyltransferases only those which present an epiarginasic regulation by their own arginase are very sensitive to the arginase inhibition in the presence of arginine. This is the case of the trimeric carbamoyl- transferase of B. subtilis [19]. To confirm the results obtained by the 'inhibition tests', molecular sieving analysis on Sephadex G-200 SF was developed to prove the possible association between the trimeric struc- tures of arginase and ornithine carbamoyltransferase. The conclusions are the same and confirm the im- portance of arginine to bring arginase into its binding conformation (Table 1) because, only when arginine is present, are the trimeric ornithine carbamoyl- transferases shifted to the higher molecular weight.

Epiarginasic Regulation in S. cerevisiae

K 10

O C I 10 ve

Fig. 6. Heterologous interaction between arginase of' S . cerevisiae and ornithine carbamoyltransferasr of E . coli W. Molecular sieving on Sephadex G-200 (0.9 x 12-cm column) at pH 8.0 in 50 mM Trisimaleate buffer and at 30 "C. (A) 0.593 nmol ornithine carba- moyltransferase with 1.06 nmol arginase. (B) The same, with 1 mM arginine. (C) The same, with 1 mM arginine, 20 mM ornithine and 20 mM citrulline. (D) 2.06 nmol ornithine carbamoyltransferase with 1.06 nmol arginase and 1 mM arginine

Further evidence is that, omitting the case of S. cere- visiue carbamoyltransferase, ornithine and citrulline do not modify the patterns on molecular sieving as shown by the study carried out with the ornithine carbamoyltransferases from E. coli (Fig. 6) . In this peculiar case the equilibrium is only modified by the mass law between arginase and carbamoyltransferase (see Fig. 6 B and D).

On the other hand, we tested the effect of other arginases from B. lichenijiormis and rat liver on orni- thine carbamoyltransferase from S. cerevisiae. The former arginase is a hexamer [12], the second is a trimer [4]. From these experiments Table 2 shows that this ornithine carbamoyltransferase cannot bind ar- ginases other than its own in the presence of all the effectors.

CONCLUSIONS

It is clearly established that citrulline is an effector of the 'epiarginasic regulation' of S. cerevisiae orni-

thine carbamoyltransferase, in vitro and in si tx The target of citrulline is the transferase.

This conclusion has been reached by studying the effect of citrulline on the binding of ornithine carbamoyltransferase to arginase and its action as effector of the inhibition of the transferase activity. The association promoted by citrulline is a trimolec- ular complex between one molecule of arginase and two molecules of ornithine carbamoyltransfer- ase. The dissociation constants measured by molec- ular sieving give values of 4 x and 1.8 x lo-* M, whereas inhibition studies give dissociation constants of 7.2 x lo-'' and 4.3 x lo-'' M corresponding re- spectively to the first and the second association. The discrepancy shown could be explained by a chaotropic effect of ionic strength on the associa- tion, as previously described by Penninckx and Wiame [6]. The analysis of heterologous associa- tions between arginases and ornithine carbamoyl- transferases prove that the trimeric structure of both partners is very important in the recognition phase of the association. These experiments further show that, during the epiarginasic regulation in S. cerevisiae, the ornithine carbamoyltransferase is the target of arginase when this latter is saturated at its regulatory site [ 5 ] , this saturation induces a conformational tran- sition of arginase bringing it into a structure which can recognize any trimeric ornithine carbamoyltrans- ferase. The various data enable us to conclude that the epiarginasic inhibition of ornithine carbamoyl- transferase develops through a sequential mechanism in which the binding phase precedes the inhibition phase. The binding stage is under the control of two specific effectors, arginine and citrulline, acting re- spectively on arginase and ornithine carbamoyltrans- ferase at sites distinct from their own catalytic sites. These sites are responsible for conformational tran- sitions which allow the formation of a trimolecular complex between the two enzymes. When ornithine is used instead of citrulline at the binding site of ornithine carbamoyltransferase only a bimolecular complex can be formed. This is perhaps proof that the real effector is citrulline, in the binding phase of the epiarginasic inhibition of ornithine carbamoyl- transferase, and that ornithine is the real effector of the inhibition phase which is induced by the saturation of the regulatory site on the ornithine carbamoyl- transferase when the enzyme is associated with ar- ginase.

V. Stalon is chercheur pal i f i t at the Fonds National dr la Recherche Scimtifique. This work was supported by contract 214245175 from the Fonds dr la Recherche Fondamentale CoNeciive. We are grateful to Prof. A. Pitrard and K. Broman for reading the manuscript.

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J.-P. Simon and V. Stalon, Institut de Recherches du C.E.R.I.A., Avenue Emile-Gryzon 1, B-1070 Bruxelles, Belgium