Methionine adenosyltransferase (MAT III), also known as S-adeno-sylmethionine synthetase, was purified from rat liver and crystallized. X-ray diffraction data were collected using a microfocused synchrotron radiation. The crystallization conditions were extensively optimized but final crystal size was never larger than 303 pm3. Due to their small size crystals had no detectable diffraction on either rotating anode source or the Deresbury SRS beamline 9.6 (GB). Finally, four data sets were collected on Grenoble ESRF (France) undulator microfocus beamline ID13 to resolution of 3.2-3.6 Å. Crystals belong to the cubic space group F432 with cell dimension a = 246 Å. Attempts are under way to solve the structure by molecular replacement, using recombinant MAT I rat liver structure as a search model

Dijana Matak-Vinković

Isabel Pérez-Mato

José M. Mato

Mladen Vinković

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ISSN-0011-1643CCA-2797 Original Scientific PaperMethionine Adenosyltransferase Purifiedfrom Rat LiverIsabel Pérez-Mato,a,b Dijana Matak-Vinkovi},a,c,*Mladen Vinkovi},a,d and José M. MatobaDepartment of Biochemistry, University of Cambridge,80 Tennis Court Road, Cambridge CB2 1GA, UKbDivision of Hepatology and Gene Therapy, Department of Medicine,University of Navarra, 31008 Pamplona, SpaincLaboratory of General and Inorganic Chemistry, Faculty of Science,University of Zagreb, Zvonimirova 8, 10000 Zagreb, CroatiadPLIVA d.d., Research and Development,Prilaz baruna Filipovi}a 25, 10000 Zagreb, CroatiaReceived October 31, 2000; revised October 11, 2001; accepted October 12, 2001Methionine adenosyltransferase (MAT III), also known as S-adeno-sylmethionine synthetase, was purified from rat liver and crystal-lized. X-ray diffraction data were collected using a microfocusedsynchrotron radiation. The crystallization conditions were extensi-vely optimized but final crystal size was never larger than 303 m3.Due to their small size crystals had no detectable diffraction on ei-ther rotating anode source or the Deresbury SRS beamline 9.6 (GB).Finally, four data sets were collected on Grenoble ESRF (France)undulator microfocus beamline ID13 to resolution of 3.2–3.6 Å.Crystals belong to the cubic space group F432 with cell dimensiona = 246 Å. Attempts are under way to solve the structure by molecu-lar replacement, using recombinant MAT I rat liver structure as asearch model.Key words: methionine adenosyltransferase, enzyme, microcrystals,microdiffraction.* Author to whom correspondence should be addressed. (E-mail: dijana@chem.pmf.hr)CROATICA CHEMICA ACTA CCACAA 75 (1) 247¿254 (2002)INTRODUCTIONMethionine adenosyltransferase (MAT), also known as S-adenosylme-thionine synthetase, catalyses the formation of S-adenosylmethionine (Ado-Met) from L-methionine and ATP (Scheme 1). In mammalian tissues, threeforms of MAT have been identified, as products of three genes. Gene MAT1A generates 1 subunits, MAT 2A generates 2 subunits and MAT 2B gen-erates ß subunits.1–5 MAT II, specific of extrahepatic tissues and fetal liver,is a heterotetramer formed by two 2 and two ß subunits.2,6 In the liver,MAT exists as a tetramer (MAT I) and as a dimer (MAT III) of  sub-units.1,7–11 The two liver enzymes with different kinetic properties, havingMAT III the highest metabolic capacity, which enables it to clear the me-thionine from the diet (a proccess that takes place in the liver).12 In the li-ver, S-adenosylmethionine, discovered by Cantoni13 in 1953, serves as themethyl donor for many biological reactions and also provides the propylami-ne group in the synthesis of polyamines. The formation of AdoMet, cataly-sed by MAT, occurs in a two-step reaction.14,15 First, AdoMet is formed bynucleophilic attack of the sulfur atom of methionine on the C5' atom of ATPin an SN2 reaction, with cleavage of the complete tripolyphosphate groupfrom ATP. In the second step, the tripolyphosphate chain is hydrolyzed toPPi and Pi before any product is released. MAT requires divalent metal ionssuch as Mg2+ for activity13 and is greatly activated by certain monovalentcations as K+. The regulatory role of nitric oxide (NO) has also been exten-sively studied.16 Comparisons of sequences of MAT from various sourcesEscherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, the leaf ofDianthus caryophyllus, rat liver, rat kidney, human liver and human kidneyshow that MAT is an exceptionally well-conserved enzyme through evolu-tion.17248 I. PÉREZ-MATO ET AL.--+--++- - --OH2+ +----+ +N NNNO O P O P ONH2P OO O OOH OHSCH3NH3COON NNNONH2OH OHSCH3NH3COOO P O P OOOOOO POOOO O OScheme 1.The crystal structure of MAT from E. coli was determined at 3 Å resolu-tion.17 The enzyme consists of 4 identical subunits, each of 383 amino acidresidues. Two subunits form a spherical, tight dimer. Each subunit consistsof three similar domains that are related to each other by pseudo-3-foldsymmetry. The essential divalent and monovalent metal ions lie in a large,deep active site cleft. Recently, the structure of the recombinant rat livertetrameric MAT I was solved at 2.7 Å in tetragonal space group P4122.18The recombinant form of the enzyme purified from inclusion bodies was se-lected because attempts to crystallize the liver purified protein were unsuc-cessful.Our objective is to solve the 3-D structure of dimeric MAT III purifiedfrom rat liver by molecular replacement, using the known structure of thetetrameric recombinant rat liver enzyme as a model.EXPERIMENTALPurificationMAT III isozyme was purified as described previously by Cabrero1 with some mi-nor modifications. The livers of 20 male rats of about 250 g were removed and placedin 300 ml of ice-cold buffer containing 0.3 mol dm–3 sucrose, 0.1 mol dm–3 EGTA, and10 mmol dm–3 Tris/HCl, pH = 7.4. Just before homogenization, benzamidine, phenyl-methylsulfonyl fluoride, and 2-mercaptoethanol were added to final concentrationsof 1 mmol dm–3, 0.1 mmol dm–3, and 0.1% (vol. fraction, ), respectively. The homoge-nate was clarified by centrifugation at 100 000 × g for 90 min. The supernatant wasloaded onto a DEAE-Sepharose Fast Flow column and equilibrated in buffer A (10mmol dm–3 MgSO4, 1 mmol dm–3 EDTA, and 10 mmol dm–3 Hepes, pH = 7.5). The en-zyme was eluted with a linear gradient of KCl in the same buffer. A single peak ofAdoMet synthetase activity eluted at about 220 mmol dm–3 KCl. Fractions contain-ing activity were pooled and loaded onto a phenyl-Sepharose equilibrated in buffer Acontaining 200 mmol dm–3 KCl. After washing the column first with equilibrationbuffer and then with buffer A, MAT III was eluted with buffer A containing 50% ()dimethyl sulfoxide. The eluate was dialyzed against buffer A and loaded onto aBlue-Sepharose Fast Flow column equilibrated in the same buffer. MAT III eluted inthe flowthrough. Fractions containing AdoMet synthetase activity were pooled andconcentrated under nitrogen using an Amicon Ultrafiltration cell fitted with a YM30membrane. The concentrated MAT III solution was cleared from particles and aggre-gates with a 0.22-µm filter, and then KCl and dithiothretiol (DTT) were added togive us final concentrations of 10 and 5 mmol dm–3, respectively. The final MAT IIIsolution was aliquoted, frozen in liquid nitrogen, and stored at –80 C. The proteinconcentration was determined using the Bio-Rad Protein Microassay, with bovine se-rum albumin as the standard, based on the method of Bradford.18 The purity of theenzyme, estimated by an overloaded SDS-polyacrylamide gel electrophoresis, wasover 95%. About 12 mg of protein were obtained in a typical purification.RAT LIVER METHIONINE ADENOSYLTRANSFERASE 249CrystallizationCrystals suitable for X-ray diffraction studies were grown using hanging-dropvapour diffusion method (Figure 1) and usually appeared within 4–8 weeks. 1–2 lof protein in 20 mmol dm–3 Hepes buffer pH = 7.0 were mixed with an equal volumereservoir precipitant solution containing 0.4–0.8 mol dm–3 (NH4)2SO4 and 100 mmoldm–3 MES buffer (pH = 6.2–6.4) at 19 C.In an attempt to obtain an ATP complex, some crystals were soaked in a solutioncontaining 0.8 mol dm–3 (NH4)2SO4, 100 mmol dm–3 MES buffer (pH = 6.0) and 10mmol dm–3 ATP for one hour. Similarly, to obtain an NO complex hanging drop con-taining crystals were exposed to an atmosphere of NO in an hermetic container for 6days.250 I. PÉREZ-MATO ET AL.Figure 1. Cubic micro-crystals of purified rat liver MAT III in hanging drop.Figure 2. Cubic micro-crystals of purified rat liver MAT III in cryo-loop. Approxi-mate dimensions are 303 m3.The cryo-protectant solution used for X-ray studies contained 0.5 mol dm–3(NH4)2SO4, 100 mmol dm–3 MES buffer (pH = 6.2) and 25% of ethylene glycol. Crys-tals were soaked in the cryo-protectant solution up to one minute and transferredinto the liquid nitrogen (Figure 2). In the case of the ATP soaked crystals the cryo-protectant solution contained 10 mmol dm–3 ATP as well.X-ray Diffraction ExperimentSeveral data sets were collected on the ESRF ID13 Microfocus beamline20 bothfrom native and ATP-soaked MAT crystals. Data were recorded at 100 K. The crys-tal-to-detector (MAR CCD 130 mm) distance was 230 mm and oscillation angle was0.5. The total oscillation range was 40 to 60. Images were processed and scaledusing DENZO21 and Scalepack22 package. Space-group determination was performedusing the autoindexing option in DENZO and by inspection of systematic absencesamong low resolution reflections.The crystals diffracted up to 3.0 Å (Figure 3). Details on data processing statis-tics of two native and two ATP soaked datasets are shown in Table I.RESULTS AND DISCUSSIONCrystallization trials were performed at two different temperatures (4C and 19 C) using the hanging-drop and the sitting drop vapour-diffusionRAT LIVER METHIONINE ADENOSYLTRANSFERASE 251Figure 3. Diffraction pattern from MAT micro-crystal.methods. Crystallization conditions were searched initially by the ammoni-um sulphate and Hampton crystal screens 1 and 2. The concentration of theprotein solution used into screening experiments was 13 mg/ml in 20 mmoldm–3 Hepes (pH = 7.0). Having obtained tiny cube-shaped micro-crystals ofthe enzyme from the solution containing 0.4 to 0.7 mol dm–3 (NH4)2SO4, 100mmol dm–3 MES buffer (pH = 5.8) 5 mmol dm–3 MgCl2 and 5 mmol dm–3KH2PO4, the optimal conditions for growing crystals for X-ray diffractionstudy were searched by varying the pH, protein concentration, precipitant,temperature and crystallization. The sitting drop method did not producecrystals while macro-seeding method did not increase crystal size.Extensive optimization of growth conditions allowed crystal size to beincreased, but never larger than 303 m3. Due to their small size crystalshave no detectable diffraction on either the rotating anode source or theSRS (Daresbury, UK) beamline 9.6. In test experiments at the ESRF (Gre-noble, France) beamline ID14 EH2, diffraction spots were recorded up to 5 Å(data not shown), suggesting the cubic space group F432 with cell dimen-sion a = 246 Å.Finally, data were collected on ESRF ID13 microfocus beamline.23 Fourdata sets were collected and data collection statistics are given in Table I.Data sets codenamed ATP1 and ATP3 were collected on crystals that weresoaked in 10 mmol dm–3 ATP solution for an hour while data sets codena-med Nat1 and Nat3 were collected on crystals directly transferred from cry-stallization drops to cryoprotectant solution. Crystals diffracted to resolu-tion of 3.2–3.6 Å and survived in the microbeam flux for 5 min to allow usthe collection of complete data sets for the cubic space group. Diffraction da-ta quality is not high judging from high Rmerge values shown in Table I. Cry-252 I. PÉREZ-MATO ET AL.TABLE IData statistics of the 4 datasets of MAT usedDataset: ATP1 ATP3 Nat1 Nat3No. observed reflexions 126414 124227 145377 18383No. unique reflexions 10546 7334 7972 9400Rmerge /% 17.8 20.0 21.7 18.6Rmerge (highest resol.)/% 63.6 55.7 42.1 48.3Completeness (all data)/% 94.3 95.6 93.1 89.1Completeness (highest resol.)/% 97.9 91.5 92.2 90.7Resolution/Å 3.4 3.2 3.7 3.4Estimated mosaicity/° 0.39 0.98 1.06 1.08stals show variable degrees of mosaicity. MAT crystals soaked in nitric oxidefor 6 days did not diffract at all.Attempts are under way to solve the structure by molecular replace-ment, using recombinant MAT I rat liver structure as a search model.Acknowledgements. – We are grateful to the Dr. Frank von Delft and to the staffof ESRF ID13 Microfocus beamline for help with diffraction experiments. We thankProfessor Tom Blundell for valuable suggestions and critical reading of the manu-script. The use of EMBO-Grenoble facilities is gratefully acknowledged.REFERENCES1. C. Cabrero, J. Puerta, and S. Alemany. Eur. J. Biochem. 170 (1987) 299–304.2. M. Kotb and N. M. Kredich. J. Biol. Chem. 260 (1985) 3923–3930.3. H. L. LeGros, A-B. Halim, A. M. Geller, and M. Kotb. J. Biol. Chem. 275 (2000)2359–2366.4. G. Okada, H. Teraoka, and K. Tsukada. Biochemistry 20 (1981) 934–940.5. D. M. Sullivan and J. L. Hoffman. Biochemistry 22 (1983) 1636–1641.6. S. Horikawa, J. Sasuga, K. Shimizu, H. Ozasa, and K. Tsukada. Eur. J. Biochem.184 (1989) 497–501.7. S. Horikawa, M. Ishikawa, H. Ozasa, and K. Tsukada. J. Biol. Chem. 265 (1990)13683–1368.8. S. Horikawa and K. Tsukada. Biochem. Int. 25 (1991) 81–90.9. L. Alvarez, M. Asuncion, F. Corrales, M. A. Pajares, and J. M. Mato. FEBS Lett.290 (1991) 142–146.10. L. Alvarez, F. Corrales, A. Martin-Duce, and J. M. Mato. Biochem. J. 293 (1993)481–486.11. K. Tsukada and G. Okada. Biochem. Biophys. Res. Commun. 94 (1980) 1078–1082.12. S. H. Mudd and J. R. Poole. Metabolism. 29 (1975) 707–720.13. G. L. Cantoni, J. Biol. Chem. 204 (1953) 403–416.14. S. H. Mudd. J. Biol. Chem. 237 (1962) PC1372–PC1375.15. S. H. Mudd. J. Biol. Chem. 238 (1963) 2456-2463.16. I. Perez-Mato, C. Castro, F. A. Ruiz, F. J. Corrales, and J. M. Mato, J. Biol. Chem.274 (1999) 17075–17079.17. F. Takusagawa, S. Kamitori, S. Misaki, and G. D. Markham, J. Biol. Chem. 271(1996) 136–147.18. B. Gonzalez, M. A. Pajares, J. A. Hermoso, L. Alvarez, F. Garrido, J. R. Sufrin,and J. Sanz-Aparicio, J. Mol. Biol. 00 (2000) 1–13.19. M. M. Bradford, Anal. Biochem. 72 (1976) 248–254.20. A. Perrakis, F. Cipriani, J. C. Castagna, L. Claustre, M. Burghammer, C. Riekeland S. Cusack, Acta Crystallogr., Sect. D 55 (1999) 1765–1770.21. Z. Otwinowski and W. Minor, Methods Enzymol. 276 (1997) 307–326.22. Collaborative Computational Project, Acta Crystallogr., Sect. D 50 (1994) 760–763.23. P. Engström, S. Fiedler, and C. Riekel, Rev. Sci. Instrum. 66 (1995) 1348–1350.RAT LIVER METHIONINE ADENOSYLTRANSFERASE 253SA@ETAKMetionin-adenoziltransferaza izolirana iz jetre {takoraIsabel Pérez-Mato, Dijana Matak-Vinkovi}, Mladen Vinkovi} i José M. MatoEnzim metionin-adenoziltransferaza (MAT III), tako|er poznat pod imenomS-adenozilmetioninsintetaza, izoliran je iz jetre {takora i kristaliziran. Kristalografskipodatci prikupljeni su upotrebom mikrofokusiranog izvora zra~enja na sinkrotronu.Iako su uvjeti kristalizacije bili u velikoj mjeri optimalizirani, kona~na veli~ina kri-stala nije prelazila 303 m3. Zbog malog volumena kristala nije detektirana difrak-cija rentgenskog zra~enja iz rentgenske cijevi s rotiraju}om anodom, kao ni na sta-nici 9.6 sinkrotrona SRS (Daresbury, Velika Britanija). ^etiri skupa podatakaprikupljena su do rezolucije 3.2–3.6 Å na stanici ID13 mikrofokusiranim zra~enjemundulatora na sinkrotronu ESRF (Grenoble, Francuska). Kristali pripadaju kubi~nojprostornoj grupi F432, s dimenzijama }elije a = 246 Å. U tijeku je rje{avanje kristal-ne strukture metodom molekulske zamjene, pri ~emu se kao polazni model korististruktura rekombinantne MAT I iz jetre {takora.254 I. PÉREZ-MATO ET AL.

English

Methionine Adenosyltransferase Purified from Rat Liver

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ISSN-0011-1643CCA-2797 Original Scientific PaperMethionine Adenosyltransferase Purifiedfrom Rat LiverIsabel Pérez-Mato,a,b Dijana Matak-Vinkovi},a,c,*Mladen Vinkovi},a,d and José M. MatobaDepartment of Biochemistry, University of Cambridge,80 Tennis Court Road, Cambridge CB2 1GA, UKbDivision of Hepatology and Gene Therapy, Department of Medicine,University of Navarra, 31008 Pamplona, SpaincLaboratory of General and Inorganic Chemistry, Faculty of Science,University of Zagreb, Zvonimirova 8, 10000 Zagreb, CroatiadPLIVA d.d., Research and Development,Prilaz baruna Filipovi}a 25, 10000 Zagreb, CroatiaReceived October 31, 2000; revised October 11, 2001; accepted October 12, 2001Methionine adenosyltransferase (MAT III), also known as S-adeno-sylmethionine synthetase, was purified from rat liver and crystal-lized. X-ray diffraction data were collected using a microfocusedsynchrotron radiation. The crystallization conditions were extensi-vely optimized but final crystal size was never larger than 303 m3.Due to their small size crystals had no detectable diffraction on ei-ther rotating anode source or the Deresbury SRS beamline 9.6 (GB).Finally, four data sets were collected on Grenoble ESRF (France)undulator microfocus beamline ID13 to resolution of 3.2–3.6 Å.Crystals belong to the cubic space group F432 with cell dimensiona = 246 Å. Attempts are under way to solve the structure by molecu-lar replacement, using recombinant MAT I rat liver structure as asearch model.Key words: methionine adenosyltransferase, enzyme, microcrystals,microdiffraction.* Author to whom correspondence should be addressed. (E-mail: dijana@chem.pmf.hr)CROATICA CHEMICA ACTA CCACAA 75 (1) 247¿254 (2002)INTRODUCTIONMethionine adenosyltransferase (MAT), also known as S-adenosylme-thionine synthetase, catalyses the formation of S-adenosylmethionine (Ado-Met) from L-methionine and ATP (Scheme 1). In mammalian tissues, threeforms of MAT have been identified, as products of three genes. Gene MAT1A generates 1 subunits, MAT 2A generates 2 subunits and MAT 2B gen-erates ß subunits.1–5 MAT II, specific of extrahepatic tissues and fetal liver,is a heterotetramer formed by two 2 and two ß subunits.2,6 In the liver,MAT exists as a tetramer (MAT I) and as a dimer (MAT III) of  sub-units.1,7–11 The two liver enzymes with different kinetic properties, havingMAT III the highest metabolic capacity, which enables it to clear the me-thionine from the diet (a proccess that takes place in the liver).12 In the li-ver, S-adenosylmethionine, discovered by Cantoni13 in 1953, serves as themethyl donor for many biological reactions and also provides the propylami-ne group in the synthesis of polyamines. The formation of AdoMet, cataly-sed by MAT, occurs in a two-step reaction.14,15 First, AdoMet is formed bynucleophilic attack of the sulfur atom of methionine on the C5' atom of ATPin an SN2 reaction, with cleavage of the complete tripolyphosphate groupfrom ATP. In the second step, the tripolyphosphate chain is hydrolyzed toPPi and Pi before any product is released. MAT requires divalent metal ionssuch as Mg2+ for activity13 and is greatly activated by certain monovalentcations as K+. The regulatory role of nitric oxide (NO) has also been exten-sively studied.16 Comparisons of sequences of MAT from various sourcesEscherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, the leaf ofDianthus caryophyllus, rat liver, rat kidney, human liver and human kidneyshow that MAT is an exceptionally well-conserved enzyme through evolu-tion.17248 I. PÉREZ-MATO ET AL.--+--++- - --OH2+ +----+ +N NNN O O P O P ONH2P OO O OOH OHSCH3NH3COON NNN ONH2OH OHSCH3NH3COOO P O P OOOOOO POOOO O OScheme 1.The crystal structure of MAT from E. coli was determined at 3 Å resolu-tion.17 The enzyme consists of 4 identical subunits, each of 383 amino acidresidues. Two subunits form a spherical, tight dimer. Each subunit consistsof three similar domains that are related to each other by pseudo-3-foldsymmetry. The essential divalent and monovalent metal ions lie in a large,deep active site cleft. Recently, the structure of the recombinant rat livertetrameric MAT I was solved at 2.7 Å in tetragonal space group P4122.18The recombinant form of the enzyme purified from inclusion bodies was se-lected because attempts to crystallize the liver purified protein were unsuc-cessful.Our objective is to solve the 3-D structure of dimeric MAT III purifiedfrom rat liver by molecular replacement, using the known structure of thetetrameric recombinant rat liver enzyme as a model.EXPERIMENTALPurificationMAT III isozyme was purified as described previously by Cabrero1 with some mi-nor modifications. The livers of 20 male rats of about 250 g were removed and placedin 300 ml of ice-cold buffer containing 0.3 mol dm–3 sucrose, 0.1 mol dm–3 EGTA, and10 mmol dm–3 Tris/HCl, pH = 7.4. Just before homogenization, benzamidine, phenyl-methylsulfonyl fluoride, and 2-mercaptoethanol were added to final concentrationsof 1 mmol dm–3, 0.1 mmol dm–3, and 0.1% (vol. fraction, ), respectively. The homoge-nate was clarified by centrifugation at 100 000 × g for 90 min. The supernatant wasloaded onto a DEAE-Sepharose Fast Flow column and equilibrated in buffer A (10mmol dm–3 MgSO4, 1 mmol dm–3 EDTA, and 10 mmol dm–3 Hepes, pH = 7.5). The en-zyme was eluted with a linear gradient of KCl in the same buffer. A single peak ofAdoMet synthetase activity eluted at about 220 mmol dm–3 KCl. Fractions contain-ing activity were pooled and loaded onto a phenyl-Sepharose equilibrated in buffer Acontaining 200 mmol dm–3 KCl. After washing the column first with equilibrationbuffer and then with buffer A, MAT III was eluted with buffer A containing 50% ()dimethyl sulfoxide. The eluate was dialyzed against buffer A and loaded onto aBlue-Sepharose Fast Flow column equilibrated in the same buffer. MAT III eluted inthe flowthrough. Fractions containing AdoMet synthetase activity were pooled andconcentrated under nitrogen using an Amicon Ultrafiltration cell fitted with a YM30membrane. The concentrated MAT III solution was cleared from particles and aggre-gates with a 0.22-µm filter, and then KCl and dithiothretiol (DTT) were added togive us final concentrations of 10 and 5 mmol dm–3, respectively. The final MAT IIIsolution was aliquoted, frozen in liquid nitrogen, and stored at –80 C. The proteinconcentration was determined using the Bio-Rad Protein Microassay, with bovine se-rum albumin as the standard, based on the method of Bradford.18 The purity of theenzyme, estimated by an overloaded SDS-polyacrylamide gel electrophoresis, wasover 95%. About 12 mg of protein were obtained in a typical purification.RAT LIVER METHIONINE ADENOSYLTRANSFERASE 249CrystallizationCrystals suitable for X-ray diffraction studies were grown using hanging-dropvapour diffusion method (Figure 1) and usually appeared within 4–8 weeks. 1–2 lof protein in 20 mmol dm–3 Hepes buffer pH = 7.0 were mixed with an equal volumereservoir precipitant solution containing 0.4–0.8 mol dm–3 (NH4)2SO4 and 100 mmoldm–3 MES buffer (pH = 6.2–6.4) at 19 C.In an attempt to obtain an ATP complex, some crystals were soaked in a solutioncontaining 0.8 mol dm–3 (NH4)2SO4, 100 mmol dm–3 MES buffer (pH = 6.0) and 10mmol dm–3 ATP for one hour. Similarly, to obtain an NO complex hanging drop con-taining crystals were exposed to an atmosphere of NO in an hermetic container for 6days.250 I. PÉREZ-MATO ET AL.Figure 1. Cubic micro-crystals of purified rat liver MAT III in hanging drop.Figure 2. Cubic micro-crystals of purified rat liver MAT III in cryo-loop. Approxi-mate dimensions are 303 m3.The cryo-protectant solution used for X-ray studies contained 0.5 mol dm–3(NH4)2SO4, 100 mmol dm–3 MES buffer (pH = 6.2) and 25% of ethylene glycol. Crys-tals were soaked in the cryo-protectant solution up to one minute and transferredinto the liquid nitrogen (Figure 2). In the case of the ATP soaked crystals the cryo-protectant solution contained 10 mmol dm–3 ATP as well.X-ray Diffraction ExperimentSeveral data sets were collected on the ESRF ID13 Microfocus beamline20 bothfrom native and ATP-soaked MAT crystals. Data were recorded at 100 K. The crys-tal-to-detector (MAR CCD 130 mm) distance was 230 mm and oscillation angle was0.5. The total oscillation range was 40 to 60. Images were processed and scaledusing DENZO21 and Scalepack22 package. Space-group determination was performedusing the autoindexing option in DENZO and by inspection of systematic absencesamong low resolution reflections.The crystals diffracted up to 3.0 Å (Figure 3). Details on data processing statis-tics of two native and two ATP soaked datasets are shown in Table I.RESULTS AND DISCUSSIONCrystallization trials were performed at two different temperatures (4C and 19 C) using the hanging-drop and the sitting drop vapour-diffusionRAT LIVER METHIONINE ADENOSYLTRANSFERASE 251Figure 3. Diffraction pattern from MAT micro-crystal.methods. Crystallization conditions were searched initially by the ammoni-um sulphate and Hampton crystal screens 1 and 2. The concentration of theprotein solution used into screening experiments was 13 mg/ml in 20 mmoldm–3 Hepes (pH = 7.0). Having obtained tiny cube-shaped micro-crystals ofthe enzyme from the solution containing 0.4 to 0.7 mol dm–3 (NH4)2SO4, 100mmol dm–3 MES buffer (pH = 5.8) 5 mmol dm–3 MgCl2 and 5 mmol dm–3KH2PO4, the optimal conditions for growing crystals for X-ray diffractionstudy were searched by varying the pH, protein concentration, precipitant,temperature and crystallization. The sitting drop method did not producecrystals while macro-seeding method did not increase crystal size.Extensive optimization of growth conditions allowed crystal size to beincreased, but never larger than 303 m3. Due to their small size crystalshave no detectable diffraction on either the rotating anode source or theSRS (Daresbury, UK) beamline 9.6. In test experiments at the ESRF (Gre-noble, France) beamline ID14 EH2, diffraction spots were recorded up to 5 Å(data not shown), suggesting the cubic space group F432 with cell dimen-sion a = 246 Å.Finally, data were collected on ESRF ID13 microfocus beamline.23 Fourdata sets were collected and data collection statistics are given in Table I.Data sets codenamed ATP1 and ATP3 were collected on crystals that weresoaked in 10 mmol dm–3 ATP solution for an hour while data sets codena-med Nat1 and Nat3 were collected on crystals directly transferred from cry-stallization drops to cryoprotectant solution. Crystals diffracted to resolu-tion of 3.2–3.6 Å and survived in the microbeam flux for 5 min to allow usthe collection of complete data sets for the cubic space group. Diffraction da-ta quality is not high judging from high Rmerge values shown in Table I. Cry-252 I. PÉREZ-MATO ET AL.TABLE IData statistics of the 4 datasets of MAT usedDataset: ATP1 ATP3 Nat1 Nat3No. observed reflexions 126414 124227 145377 18383No. unique reflexions 10546 7334 7972 9400Rmerge /% 17.8 20.0 21.7 18.6Rmerge (highest resol.)/% 63.6 55.7 42.1 48.3Completeness (all data)/% 94.3 95.6 93.1 89.1Completeness (highest resol.)/% 97.9 91.5 92.2 90.7Resolution/Å 3.4 3.2 3.7 3.4Estimated mosaicity/° 0.39 0.98 1.06 1.08stals show variable degrees of mosaicity. MAT crystals soaked in nitric oxidefor 6 days did not diffract at all.Attempts are under way to solve the structure by molecular replace-ment, using recombinant MAT I rat liver structure as a search model.Acknowledgements. – We are grateful to the Dr. Frank von Delft and to the staffof ESRF ID13 Microfocus beamline for help with diffraction experiments. We thankProfessor Tom Blundell for valuable suggestions and critical reading of the manu-script. The use of EMBO-Grenoble facilities is gratefully acknowledged.REFERENCES1. C. Cabrero, J. Puerta, and S. Alemany. Eur. J. Biochem. 170 (1987) 299–304.2. M. Kotb and N. M. Kredich. J. Biol. Chem. 260 (1985) 3923–3930.3. H. L. LeGros, A-B. Halim, A. M. Geller, and M. Kotb. J. Biol. Chem. 275 (2000)2359–2366.4. G. Okada, H. Teraoka, and K. Tsukada. Biochemistry 20 (1981) 934–940.5. D. M. Sullivan and J. L. Hoffman. Biochemistry 22 (1983) 1636–1641.6. S. Horikawa, J. Sasuga, K. Shimizu, H. Ozasa, and K. Tsukada. Eur. J. 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Biol. 00 (2000) 1–13.19. M. M. Bradford, Anal. Biochem. 72 (1976) 248–254.20. A. Perrakis, F. Cipriani, J. C. Castagna, L. Claustre, M. Burghammer, C. Riekeland S. Cusack, Acta Crystallogr., Sect. D 55 (1999) 1765–1770.21. Z. Otwinowski and W. Minor, Methods Enzymol. 276 (1997) 307–326.22. Collaborative Computational Project, Acta Crystallogr., Sect. D 50 (1994) 760–763.23. P. Engström, S. Fiedler, and C. Riekel, Rev. Sci. Instrum. 66 (1995) 1348–1350.RAT LIVER METHIONINE ADENOSYLTRANSFERASE 253SA@ETAKMetionin-adenoziltransferaza izolirana iz jetre {takoraIsabel Pérez-Mato, Dijana Matak-Vinkovi}, Mladen Vinkovi} i José M. MatoEnzim metionin-adenoziltransferaza (MAT III), tako|er poznat pod imenomS-adenozilmetioninsintetaza, izoliran je iz jetre {takora i kristaliziran. Kristalografskipodatci prikupljeni su upotrebom mikrofokusiranog izvora zra~enja na sinkrotronu.Iako su uvjeti kristalizacije bili u velikoj mjeri optimalizirani, kona~na veli~ina kri-stala nije prelazila 303 m3. Zbog malog volumena kristala nije detektirana difrak-cija rentgenskog zra~enja iz rentgenske cijevi s rotiraju}om anodom, kao ni na sta-nici 9.6 sinkrotrona SRS (Daresbury, Velika Britanija). ^etiri skupa podatakaprikupljena su do rezolucije 3.2–3.6 Å na stanici ID13 mikrofokusiranim zra~enjemundulatora na sinkrotronu ESRF (Grenoble, Francuska). Kristali pripadaju kubi~nojprostornoj grupi F432, s dimenzijama }elije a = 246 Å. U tijeku je rje{avanje kristal-ne strukture metodom molekulske zamjene, pri ~emu se kao polazni model korististruktura rekombinantne MAT I iz jetre {takora.254 I. PÉREZ-MATO ET AL.

Methionine Adenosyltransferase Purified from Rat Liver

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