J. Mol. Biol. (1990) 215, 341-344
Trigonal Crystals of Porcine Mitochondrial Aspartate Aminotransferase Tina Izard, Bruno Fol, Richard A. Pauptit and Johan N. Jansordus Department of Structural Biology Biocentre, University of Basel Klingelbergstrasse 70 CH-4056 Basel, Switzerland
(Received 6 April 1990; accepted 19 June 1990) Crystals suitable for X-ray analysis of porcine mitochondrial aspartate aminotransferase in the closed conformation were obtained after the apoenzyme was reconstituted with _hr-5'-phosphopyridoxyl-L-aspartate, an inhibitor in which the cofactor is covalently bound to the substrate. This results in a crystal form that has not been encountered previously in studies of aspartate aminotransferases. The crystals belong to the trigonal space group P3121 (or the enantiomeric P3221) with unit cell dimensions a=b=202"0A, c=58"0 A, a = f l = 9 0 ~ ? = 120 ~ and contain one dimer in the asymmetric unit.
AATase, see Braunstein (1973) and Christen & Metzler (1985). For the last 15 years, studies on three-dimensional structure and structure-function relationships of AATases have been carried out by X-ray crystallography. Table 1 lists the crystal data that have been published to date by various laboratories. The polypeptide fold initially determined for chicken mAATase (Ford et al., 1980) has subsequently been confirmed for chicken cAATase (Borisov et al., 1985; Harutyunyan et al., 1985), pig cAATase (Arnone et at., 1985), E. coli AATase (Smith et al., 1989; Kamitori et al., 1988; J/iger et al., 1990) and, most recently, for pig mAATase (G. Pfliigl et al., unpublished results). The two subunits are related by a molecular 2fold axis. They consist of a large PEP-binding domain and a fexibly hinged small domain. The active sites lie at the subunit interface, with contributions from residues from both subunits. Accordingly, the AATase monomer is inactive (Arrio-Dupont & Coulet, 1979). In the unliganded enzyme, the active site is open to solvent (open conformation). The presence of substrate analogues such as maleate induces reorientation of the small domain to form the closed conformation, in which the active site and inhibitor are completely enclosed within the protein matrix, inaccessible to solvent. Nearly all active site residues are fully conserved, indicating that the catalytic mechanism (Kirsch et al., 1984) must be the same for eAATases, mAATases and E. coli AATase. For an extensive review on structure and function of AATase, see
Aspartate aminotransferase (AATaset) is a pyridoxal phosphate (PLP) dependent a 2 dimerie enzyme of subunit Mr around 45,000 and about 400 amino acid residues per polypeptide chain. It is the most thoroughly studied representative of the large family of vitamin B6-dependent enzymes functioning in amino acid metabolism and catalyses the reversible reaction: L-aspartate + 2-oxoglutarate ~-oxaloacetate + L-glutamate The reaction proceeds through a series of known covalent intermediates, several of which can be distinguished by their eharaeteristie ultraviolet light absorption and e{reular diehroism spectra. In higher organisms, two isoenzymes of AATase are found, one in tile eytosol (eAATase), the other in the mitochondria (mAATase). They are encoded by separate genes. The amino acid sequences for many AATases are known. Typieally, eAATases as a group and mAATases as a group have about 85~/o sequence identity, while the sequences of the eAATase and mAATase of the same species are, on average, 47~/o identical. Escherichia coli AATase shows about 40~o sequence identity with cAATases and mAATases (Mehta et al., 1989). For reviews on the bioehemieal and biophysical pr.operties of J" Abbreviations used: AATase, aspartate aminotransferase; PLP, pyridoxal-5'-phosphat'e; eAATase, eytosolie aspartate aminotransferase; mAATase, mitochondrial aspartate aminotransferase; PPL-Asp, N-5'-phosphopyridoxyl-L-aspartate. 0022-2836190/19o341-04 $03.00/0
341
1990 AcademicPress Limited
342
T . l z a r d et al. Table
1
C r y s t a l f o r m s o f A A T a s e studied to date
AATase
Space group
Cell parameterst
I'll
N~
References
A. cAATases
Chicken (closed) Pig (open)
P212121 P212121
62"7, 118"1, 124"5(A) 124.7, 130-9,55-7 (A)
2"5 2"46
2 2
Borisovet at., 1978 Arnoneet al., 1977
Chicken (open)
PI
2"3
2
Gehringel at., 1977
Pig (open)
PI
2-3
2
Eiehele*t al., 1979
Ox (open)
PI
2-3
2
Capassoet al., 1979
Chicken (closed)
C2221
55"6, 58"7, 76-0(A) 85"3, 109.2, 115.6(~ 55"9, 58"5, 77-0(A) 85"0, 109.8, 115.9 (o) 55"6, 58"6, 76"0(A) 85"5, 109'l, 115.6(~ 69-7, 91-4, 128-5(A)
2-3
1
Chicken (closed)
p21
Pig (closed)
P3121 or P3221
B. mAATases
57-1, 52-2, 136-3(A) 90, 101"4,90 (o) 202.0, 202-0, 58"0(A) 90, 90, 120 (~
2.2
2
Jansonius & Vincent, 1987; Jansonius tt al., 1987 Thaller ~t al., 1981
3"75
2
This work
156, 87-6, 80"6(A) 157"1, 8-3"5,79"7(A) 86"8, 79"9, 89-4(A) 90, 118'74, 90(~
3"2 3'1 3"15
1 1 2
Smith el at., 1986 Kamitori el al., 1987 J/iger et al., 1989
C. Bacterial AATases
E. coli ("half open") E. colt (elosed.~w E. colt (closed)
C2221 C2221 1'21
~"Angles not given are 90~ Number of subunits per asymmetric unit. wNot mentioned in the literature but presumed from crystallization conditions.
Jansonius & Vincent (1987). The roles of specific amino acids are n o w being examined through crystal structure determinations of m u t a n t E . colt AATases (Smith el al., 1989; J//ger et al., 1990). So far, there is no structure for porcine mAATase in the closed conformation. With the aim of allowing tire comparison of both open and closed structures of chicken and pig mAATase, co-crystallization experiments of pig mAATase with maleate have been carried out. Similar experiments resulted in well-diffracting crystals of the closed structure of chicken mAATase (Picot, 1987). However, no suitable crystals of pig mAATase could be grown in this way. Therefore, it was" decided to bind tile coenzyme-substrate analogue N-5'-l)hosphopyridoxy l_ L-asl)artate (PPL-Asl)). to tim al)oenzyme. This should stabilize tim cl(]se(l conformation of pig mAATase. At the same time, it would provide an excellent model for the so-called external aidimine intermediate in catalysis (the tirst covalent coenzyme substrate intermediate). So far in high-resolution X-ray studies, tile inhibitor 2-methylaspartate had been used to mimic this intermediate (Jansonius & Vincent, 1987). The 2-methyl group, however, might cause a local eonformational change and/or displace a possil)ly fimetiona] w.ater molecule. Thus, iml)ortant new information on the active site structure of AATase could now be gained. Here, we present the l)reI)aration, erTstallization and t)reliminat T X-ray characterization of the closed form of porcine mAATase. Pig heart mAATase was isolated as described by
Glatthaar et al. (1974), but tile purification of tile enzyme was improved in our laboratory by replacing the final two gel filtrations by a Blue-Sel)harose affinity column, following tile Bhm-Sepharose synthesis method of Lowe & Pearson (1984). The enzyme was incubated with 50 m.~t-sodium phosphate (pH 7"5), 20 m3t-cysteinesulphinic acid for 15 minutes at 4~ Within 24 hours, tile protein solution was dialysed three times against 0.5 .~I-sodium phosI)hate (t)H5"0) and twice against 20m3t3-morI)holino-I)ropanesulphonic acid. Tile resulting aI)oenzyme was reconstituted with PPL-Asp, which was synthesized according to the procedure of Severin et al. (1969). The l)rotein concentration was estimated from its absorbance at 280 nm (using a molar absorptivity of 126,000 .~x-lcm -1 for a dinaer) to be 32"3 mg/ml. Crystals were grown at 4~ by the sitting-drop vapour diffusion method, using muitiwell tissueculture plates. "File protein compartments contained 70 Id of 7"0 nag protein/ml, 11 ~/o (w/v) polyethylene glycol with a molecular weight of 4000 (PEG 4000) as precipitating agent, and 50 m.~t-potassium phosphate (I)H 7"5). The wells were equilibrated with 10 mi of reservoir solution containing 2 2 % (w/v) PEG 4000 and the same buffer in a Petri dish. Colourless crystals appeared within a few days and grew to sizes of 3 mm • 0"15 m m x 0-15 nun in three or four weeks. Tile clTstais were characterized by precession photography, using CuKa radiation from a rotating anode generator. The unit cell dimensions are
Communications
a = b = 202"0 A, c = 5 8 " 0 A (1 A=0-1 nm), a = f l = 9 0 ~ and ~,= 120 ~ The c-axis corresponds to the crystal needle axis. The presence of six dimcrs (91,000 Da per dimer) in the unit cell would give a volume to mass ratio (VM) of 3"75 A3/Da, which is equivalent to a solvent content of 67~/o and represents a loose packing for proteins (Matthews, 1968). The crystals diffract to 2.4 A. Whereas the zero-level hkO precession photograph had 6mm symmetry, the upper-level h k l photograph showcd 3m s y m m e t r y with the mirror planes every 60 ~ about c*, along a* and b*. This indicates the presence of 2-fold axes perpendicular to and related by a 3-fold axis. The 2-fold axes lie normal to the mirror planes, i.e., along the real axial directions. Tim systematic absences along c* (only the 0,0,6 reflection was observed) indicate that the 3-fold is a screw axis. The hO1 and 0kl precession photographs show only Friedel symmetry, whereas the diagonal hhl zone has mm symmetry. Thus the crystals belong to the trigonal space group P3121 (or its enantiomer P3221), in which the 2-fold axes lie in ttm directions of the real axes a and b. This is the first time trigonal crystals have been found for an aspartate aminotransferase. The space group contains six asymmetric units per unit cell, implying t h a t each asymmetric unit must contain a dimer. The short c dimension suggests t h a t the dimer, with expected dimensions of about 100 A x 60 A x 50 A, probably lies with its long axis near the ab plane, This work was made possible through Swiss National Science Foundation grant 31-25713.88 to J.N.J. References Arnone, A., Rogers, P. H., Schmidt, J., ttan, C.-H., Harris, C. M. & Metzler, D. E. (1977). Preliminary Crystallographic Study of Aspartate: 2-Oxoglutarate Aminotransfcrase from Pig Heart. J. Mol. Biol. 112, 509-513. Arnone, A., Rogers, P. H., Hyde, C. C., Briley, P. D., Metzler, C. M. & Metzler, D. E. (1985). Pig Cytosolie Aspartate Aminotransferase: The Structures of the Internal Aldimine, E~ternal AIdimine, and Ketimine and of the fl Subform. In Transaminases (Christen, P. & Metzler, D. E., eds), pp. 138-155, John Wiley & Sons, New York. Arrio-Dupont, M. & Coulet, P. R. (1979). Aspartate Aminotransferase Immobilized on Collagen Fibns: Activity of Dissociated Subunits. Biochem. Biophys. Res. Commun. 89, 345-352. Borisov, V. V., Borisova, S. N., Kachalova, G. S., Sosfenov, N. I., Vainshtein, B. K., Torchinsky, Y. M. & Braunstein, A. E. (1978). Three-dimensional Structure at 5 A Resolution of Cytosolic Aspartatc Transaminase from Chicken Heart. J. Mol. Biol. 125, 275-292. Borisov, V. V., Borisova, S. N., Kachal6va, G. S., Sosfenov, N. I. & Vainshtein, B. K. (1985). X-ray Studies of Chicken Cytosolie Aspartate Aminotransferase. In Transaminases (Christen, P. & Metzler, D. E., eds), pp. 155-164, John Wiley & Sons, New York. : Braunstein, A. E. (1973). Amino Group Transfer. In The
343
Enzymes (Boyer, P. D., ed.), 3rd edit., vol. 9, pp.
379-481, Academic Press, New York. Capasso, S., Garzillo, A. M., Marino, G., Mazzarella, L., Pucci, P. & Sannia, G. (1979). Mitochondrial Bovine Aspartate Aminotransferase. F E B S Letters, 101, 351-354. Christen, P. & Metzler, D. E. (1985). Editors of Trans'aminases, John Wiley & Sons, New York. Eichele, G., Ford, G. C. & Jansonius, J. N. 0979). Crystallization of Pig Mitochondrial Aspartate Aminotransferase by Seeding with Crystals of the Chicken Mitochondrial Isoenzyme. J. Mol. Biol. 135, 513-516. Ford, G. C., Eichele, G. & Jansonius, J. N. (1980). Three-Dimensional Structure of a PyridoxalPhosphate-Dependent Enzyme, Mitochondrial Aspartate Aminotransferase. Proc. Nat. Acad. Sci., U.S.A. 77, 2559--2563. Gehring, H., Christen, P., Eichele, G., Glor, M., Jansonius, J. N., Reimer, A.-S., Smit, J. D. G. & Thaller, C. (1977). Isolation, Crystallization and Preliminary Crystallographic Data of Aspartate Aminotransferase from Chicken Heart Mitochondria. J. Mol. Biol. 115, 97-101. Glatthaar, B. E., Barbarash, G. R., Noyes, B. E., Banaszak, L. J. & Bradshaw, R. A. (1974). The Preparation of the Cytoplasmic and Mitochondrial Forms of Malate Dehydrogenase and Aspartate Aminotransferase from Pig Heart by a Single Procedure Anal. Biochem. 57, 432-451. Harutyunyan, E. G., Malasbkevich, V. N., Kochkina, V. M. & Torchinsky, Y. M. (1985). ThreeDimensional Structure of the Complex of Chicken Cytosolic Aspartate Aminotransferase with 2-oxoglutarate. In Transaminases (Christen, P. & Metzler, D. E., eds), pp. 164-173, John Wiley & Sons, New York. Jiiger, J., KShler, E., Tucker, P., Sander, U., Housley-Markovic, Z., Fotheringham, I., Edwards, M., Hunter, M., Kirschner, K. & Jansonius, J. N. (1989). Crystallization and Preliminary X-ray Studies of an Aspartate Aminotransferase Mutant from Eseherichia coll. J. Mol. Biol. 209, 499-501. J~iger, J., Moser, M., Pauptit, R., Sauder, U., Tucker, P., K6hler, E., Seville, M., Christen, P., Kirschner, K. & Jansonius, J. (1990). X-ray Studies of Mutants and Inhibitor Complexes of Aspartate Aminotransferase from E. coll. Experientia, 46, A35. Jansonius, J. N. & Vincent, M. G. (1987). Structural Basis for Catalysisby Aspartate Aminotransferase. In Biological Macromolecules d: Assemblies (Jurnak, F. A. & McPherson, A., eds), vol. 3, Active Sites of Enzymes, pp. 187-285, John Wiley & Sons, New York. Jansonius, J. N., Vincent, M. G., McPhalen, C. A. & Picot, D. (1987). Spatial Aspects of Catalysis by Mitochondrial Aspartate Aminotransferase. In Biochemistry of Vitamin B 6 (Korpela, T. & Christen, P., eds), pp. 89-98, Birkh~iuser, Basel. Kamitori, S., Hirotsu, K., Higuchi, T., Kondo, K., Inoue, K., Kuramitsu, S., Kagamiyama, H., Higuchi, Y., Yasuoka, N., Kusunoki, M. & Matsuura, Y. (1987). Overproduction and Preliminary X-ray Characterization of Aspartate Aminotransfcrase from Escherichia coli. J. Biochem. (Tokyo), 101,813-816. Kamitori, S., Hirotsu, K., Higucbi, T., Kondo, K., ]noue, K., Katsura, S., Kuramitsu, S., Kagamiyama, H., Higuchi, Y., Yasuoka, N., Kusunoki, M. & Matsuura, Y. (1988). Three-Dimensional Structure of Aspartate
344
T. Izard et al.
Aminotransferase from Escherichia coli at 2-8A Resolution. J. Biochem. (Tokyo), 104, 317-318. Kitsch, J. F., Eiehele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H. & Christen, P. (1984). Mechanism of Action of Aspartate Aminotransferase Proposed on the Basis of its Spa:ial Structure. J. Mol. Biol. 174, 497-525. Lowe, C. R. & Pearson, J. C. (1984). Affinity Chromatography on Immobilized Dyes. Methods Enzymol. 104, 97-113. Matthews, B. W. (1968). Solvent Content of Protein Crystals. J. Mol. Biol. 33,491-497. Mehta, P. K., Hale, T. I. & Christen, P. (1989). Evolutionary Relationships Among Aminotransferases: Tyrosine Aminotransferase, Histidinol Phosphate Aminotransferase, and Aspartate Aminotransferase are Homologous Proteins. Eur. J. Biochem. 186, 249-253. Picot, /). (1987). Conformational changes of Mitochondrial Aspartate Aminotransferase during
Catalysis: An X-ray Crystallographic and Microspectrophotometrie Study. Ph.D thesis, University of Basel. Severin, E. S., Gulyaev, N. N., Khurs, E. N. & Khomutov, R. M. (1969). The Synthesis and Properties of Phosphopyridoxyl Amino Acids. Biochem. Biophys. Res. Commun. 35, 318--323. Smith, D. L., Ringe, D., Finlayson, W. L. & Kirsch, J. F. (1986}. Preliminary X-ray Data for Aspartate Aminotransferase from Escherichia coll. J. Mol. Biol. 191,301-302. Smith, D. L., Almo, S. C., Toney, M. D. & Ringe, D. (1989). 2"8A Resolution Crystal Structure of an Active Site Mutant of Aspartate Aminotransferase from Escherichia coli. Biochemistry, 28, 8161-8167. Thaller, C., Weaver, L. H., Eichele, G., Wilson, E., Karlsson, R. & Jansonius, J. N. (1981}. Repeated Seeding Technique for Growing Large Single Crystals of Proteins. J. Mol. Biol. 147, 465-469.
Edited by R. Huber
Note added in proof. The structure has been solved by molecular replacement. The space group is P3121. Rings of 12 dimers surround the 3t-axis forming solvent channels of 130 A diameter.
Trigonal Crystals of Porcine Mitochondrial Aspartate Aminotransferase Tina Izard, Bruno Fol, Richard A. Pauptit and Johan N. Jansordus Department of Structural Biology Biocentre, University of Basel Klingelbergstrasse 70 CH-4056 Basel, Switzerland
(Received 6 April 1990; accepted 19 June 1990) Crystals suitable for X-ray analysis of porcine mitochondrial aspartate aminotransferase in the closed conformation were obtained after the apoenzyme was reconstituted with _hr-5'-phosphopyridoxyl-L-aspartate, an inhibitor in which the cofactor is covalently bound to the substrate. This results in a crystal form that has not been encountered previously in studies of aspartate aminotransferases. The crystals belong to the trigonal space group P3121 (or the enantiomeric P3221) with unit cell dimensions a=b=202"0A, c=58"0 A, a = f l = 9 0 ~ ? = 120 ~ and contain one dimer in the asymmetric unit.
AATase, see Braunstein (1973) and Christen & Metzler (1985). For the last 15 years, studies on three-dimensional structure and structure-function relationships of AATases have been carried out by X-ray crystallography. Table 1 lists the crystal data that have been published to date by various laboratories. The polypeptide fold initially determined for chicken mAATase (Ford et al., 1980) has subsequently been confirmed for chicken cAATase (Borisov et al., 1985; Harutyunyan et al., 1985), pig cAATase (Arnone et at., 1985), E. coli AATase (Smith et al., 1989; Kamitori et al., 1988; J/iger et al., 1990) and, most recently, for pig mAATase (G. Pfliigl et al., unpublished results). The two subunits are related by a molecular 2fold axis. They consist of a large PEP-binding domain and a fexibly hinged small domain. The active sites lie at the subunit interface, with contributions from residues from both subunits. Accordingly, the AATase monomer is inactive (Arrio-Dupont & Coulet, 1979). In the unliganded enzyme, the active site is open to solvent (open conformation). The presence of substrate analogues such as maleate induces reorientation of the small domain to form the closed conformation, in which the active site and inhibitor are completely enclosed within the protein matrix, inaccessible to solvent. Nearly all active site residues are fully conserved, indicating that the catalytic mechanism (Kirsch et al., 1984) must be the same for eAATases, mAATases and E. coli AATase. For an extensive review on structure and function of AATase, see
Aspartate aminotransferase (AATaset) is a pyridoxal phosphate (PLP) dependent a 2 dimerie enzyme of subunit Mr around 45,000 and about 400 amino acid residues per polypeptide chain. It is the most thoroughly studied representative of the large family of vitamin B6-dependent enzymes functioning in amino acid metabolism and catalyses the reversible reaction: L-aspartate + 2-oxoglutarate ~-oxaloacetate + L-glutamate The reaction proceeds through a series of known covalent intermediates, several of which can be distinguished by their eharaeteristie ultraviolet light absorption and e{reular diehroism spectra. In higher organisms, two isoenzymes of AATase are found, one in tile eytosol (eAATase), the other in the mitochondria (mAATase). They are encoded by separate genes. The amino acid sequences for many AATases are known. Typieally, eAATases as a group and mAATases as a group have about 85~/o sequence identity, while the sequences of the eAATase and mAATase of the same species are, on average, 47~/o identical. Escherichia coli AATase shows about 40~o sequence identity with cAATases and mAATases (Mehta et al., 1989). For reviews on the bioehemieal and biophysical pr.operties of J" Abbreviations used: AATase, aspartate aminotransferase; PLP, pyridoxal-5'-phosphat'e; eAATase, eytosolie aspartate aminotransferase; mAATase, mitochondrial aspartate aminotransferase; PPL-Asp, N-5'-phosphopyridoxyl-L-aspartate. 0022-2836190/19o341-04 $03.00/0
341
1990 AcademicPress Limited
342
T . l z a r d et al. Table
1
C r y s t a l f o r m s o f A A T a s e studied to date
AATase
Space group
Cell parameterst
I'll
N~
References
A. cAATases
Chicken (closed) Pig (open)
P212121 P212121
62"7, 118"1, 124"5(A) 124.7, 130-9,55-7 (A)
2"5 2"46
2 2
Borisovet at., 1978 Arnoneet al., 1977
Chicken (open)
PI
2"3
2
Gehringel at., 1977
Pig (open)
PI
2-3
2
Eiehele*t al., 1979
Ox (open)
PI
2-3
2
Capassoet al., 1979
Chicken (closed)
C2221
55"6, 58"7, 76-0(A) 85"3, 109.2, 115.6(~ 55"9, 58"5, 77-0(A) 85"0, 109.8, 115.9 (o) 55"6, 58"6, 76"0(A) 85"5, 109'l, 115.6(~ 69-7, 91-4, 128-5(A)
2-3
1
Chicken (closed)
p21
Pig (closed)
P3121 or P3221
B. mAATases
57-1, 52-2, 136-3(A) 90, 101"4,90 (o) 202.0, 202-0, 58"0(A) 90, 90, 120 (~
2.2
2
Jansonius & Vincent, 1987; Jansonius tt al., 1987 Thaller ~t al., 1981
3"75
2
This work
156, 87-6, 80"6(A) 157"1, 8-3"5,79"7(A) 86"8, 79"9, 89-4(A) 90, 118'74, 90(~
3"2 3'1 3"15
1 1 2
Smith el at., 1986 Kamitori el al., 1987 J/iger et al., 1989
C. Bacterial AATases
E. coli ("half open") E. colt (elosed.~w E. colt (closed)
C2221 C2221 1'21
~"Angles not given are 90~ Number of subunits per asymmetric unit. wNot mentioned in the literature but presumed from crystallization conditions.
Jansonius & Vincent (1987). The roles of specific amino acids are n o w being examined through crystal structure determinations of m u t a n t E . colt AATases (Smith el al., 1989; J//ger et al., 1990). So far, there is no structure for porcine mAATase in the closed conformation. With the aim of allowing tire comparison of both open and closed structures of chicken and pig mAATase, co-crystallization experiments of pig mAATase with maleate have been carried out. Similar experiments resulted in well-diffracting crystals of the closed structure of chicken mAATase (Picot, 1987). However, no suitable crystals of pig mAATase could be grown in this way. Therefore, it was" decided to bind tile coenzyme-substrate analogue N-5'-l)hosphopyridoxy l_ L-asl)artate (PPL-Asl)). to tim al)oenzyme. This should stabilize tim cl(]se(l conformation of pig mAATase. At the same time, it would provide an excellent model for the so-called external aidimine intermediate in catalysis (the tirst covalent coenzyme substrate intermediate). So far in high-resolution X-ray studies, tile inhibitor 2-methylaspartate had been used to mimic this intermediate (Jansonius & Vincent, 1987). The 2-methyl group, however, might cause a local eonformational change and/or displace a possil)ly fimetiona] w.ater molecule. Thus, iml)ortant new information on the active site structure of AATase could now be gained. Here, we present the l)reI)aration, erTstallization and t)reliminat T X-ray characterization of the closed form of porcine mAATase. Pig heart mAATase was isolated as described by
Glatthaar et al. (1974), but tile purification of tile enzyme was improved in our laboratory by replacing the final two gel filtrations by a Blue-Sel)harose affinity column, following tile Bhm-Sepharose synthesis method of Lowe & Pearson (1984). The enzyme was incubated with 50 m.~t-sodium phosphate (pH 7"5), 20 m3t-cysteinesulphinic acid for 15 minutes at 4~ Within 24 hours, tile protein solution was dialysed three times against 0.5 .~I-sodium phosI)hate (t)H5"0) and twice against 20m3t3-morI)holino-I)ropanesulphonic acid. Tile resulting aI)oenzyme was reconstituted with PPL-Asp, which was synthesized according to the procedure of Severin et al. (1969). The l)rotein concentration was estimated from its absorbance at 280 nm (using a molar absorptivity of 126,000 .~x-lcm -1 for a dinaer) to be 32"3 mg/ml. Crystals were grown at 4~ by the sitting-drop vapour diffusion method, using muitiwell tissueculture plates. "File protein compartments contained 70 Id of 7"0 nag protein/ml, 11 ~/o (w/v) polyethylene glycol with a molecular weight of 4000 (PEG 4000) as precipitating agent, and 50 m.~t-potassium phosphate (I)H 7"5). The wells were equilibrated with 10 mi of reservoir solution containing 2 2 % (w/v) PEG 4000 and the same buffer in a Petri dish. Colourless crystals appeared within a few days and grew to sizes of 3 mm • 0"15 m m x 0-15 nun in three or four weeks. Tile clTstais were characterized by precession photography, using CuKa radiation from a rotating anode generator. The unit cell dimensions are
Communications
a = b = 202"0 A, c = 5 8 " 0 A (1 A=0-1 nm), a = f l = 9 0 ~ and ~,= 120 ~ The c-axis corresponds to the crystal needle axis. The presence of six dimcrs (91,000 Da per dimer) in the unit cell would give a volume to mass ratio (VM) of 3"75 A3/Da, which is equivalent to a solvent content of 67~/o and represents a loose packing for proteins (Matthews, 1968). The crystals diffract to 2.4 A. Whereas the zero-level hkO precession photograph had 6mm symmetry, the upper-level h k l photograph showcd 3m s y m m e t r y with the mirror planes every 60 ~ about c*, along a* and b*. This indicates the presence of 2-fold axes perpendicular to and related by a 3-fold axis. The 2-fold axes lie normal to the mirror planes, i.e., along the real axial directions. Tim systematic absences along c* (only the 0,0,6 reflection was observed) indicate that the 3-fold is a screw axis. The hO1 and 0kl precession photographs show only Friedel symmetry, whereas the diagonal hhl zone has mm symmetry. Thus the crystals belong to the trigonal space group P3121 (or its enantiomer P3221), in which the 2-fold axes lie in ttm directions of the real axes a and b. This is the first time trigonal crystals have been found for an aspartate aminotransferase. The space group contains six asymmetric units per unit cell, implying t h a t each asymmetric unit must contain a dimer. The short c dimension suggests t h a t the dimer, with expected dimensions of about 100 A x 60 A x 50 A, probably lies with its long axis near the ab plane, This work was made possible through Swiss National Science Foundation grant 31-25713.88 to J.N.J. References Arnone, A., Rogers, P. H., Schmidt, J., ttan, C.-H., Harris, C. M. & Metzler, D. E. (1977). Preliminary Crystallographic Study of Aspartate: 2-Oxoglutarate Aminotransfcrase from Pig Heart. J. Mol. Biol. 112, 509-513. Arnone, A., Rogers, P. H., Hyde, C. C., Briley, P. D., Metzler, C. M. & Metzler, D. E. (1985). Pig Cytosolie Aspartate Aminotransferase: The Structures of the Internal Aldimine, E~ternal AIdimine, and Ketimine and of the fl Subform. In Transaminases (Christen, P. & Metzler, D. E., eds), pp. 138-155, John Wiley & Sons, New York. Arrio-Dupont, M. & Coulet, P. R. (1979). Aspartate Aminotransferase Immobilized on Collagen Fibns: Activity of Dissociated Subunits. Biochem. Biophys. Res. Commun. 89, 345-352. Borisov, V. V., Borisova, S. N., Kachalova, G. S., Sosfenov, N. I., Vainshtein, B. K., Torchinsky, Y. M. & Braunstein, A. E. (1978). Three-dimensional Structure at 5 A Resolution of Cytosolic Aspartatc Transaminase from Chicken Heart. J. Mol. Biol. 125, 275-292. Borisov, V. V., Borisova, S. N., Kachal6va, G. S., Sosfenov, N. I. & Vainshtein, B. K. (1985). X-ray Studies of Chicken Cytosolie Aspartate Aminotransferase. In Transaminases (Christen, P. & Metzler, D. E., eds), pp. 155-164, John Wiley & Sons, New York. : Braunstein, A. E. (1973). Amino Group Transfer. In The
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379-481, Academic Press, New York. Capasso, S., Garzillo, A. M., Marino, G., Mazzarella, L., Pucci, P. & Sannia, G. (1979). Mitochondrial Bovine Aspartate Aminotransferase. F E B S Letters, 101, 351-354. Christen, P. & Metzler, D. E. (1985). Editors of Trans'aminases, John Wiley & Sons, New York. Eichele, G., Ford, G. C. & Jansonius, J. N. 0979). Crystallization of Pig Mitochondrial Aspartate Aminotransferase by Seeding with Crystals of the Chicken Mitochondrial Isoenzyme. J. Mol. Biol. 135, 513-516. Ford, G. C., Eichele, G. & Jansonius, J. N. (1980). Three-Dimensional Structure of a PyridoxalPhosphate-Dependent Enzyme, Mitochondrial Aspartate Aminotransferase. Proc. Nat. Acad. Sci., U.S.A. 77, 2559--2563. Gehring, H., Christen, P., Eichele, G., Glor, M., Jansonius, J. N., Reimer, A.-S., Smit, J. D. G. & Thaller, C. (1977). Isolation, Crystallization and Preliminary Crystallographic Data of Aspartate Aminotransferase from Chicken Heart Mitochondria. J. Mol. Biol. 115, 97-101. Glatthaar, B. E., Barbarash, G. R., Noyes, B. E., Banaszak, L. J. & Bradshaw, R. A. (1974). The Preparation of the Cytoplasmic and Mitochondrial Forms of Malate Dehydrogenase and Aspartate Aminotransferase from Pig Heart by a Single Procedure Anal. Biochem. 57, 432-451. Harutyunyan, E. G., Malasbkevich, V. N., Kochkina, V. M. & Torchinsky, Y. M. (1985). ThreeDimensional Structure of the Complex of Chicken Cytosolic Aspartate Aminotransferase with 2-oxoglutarate. In Transaminases (Christen, P. & Metzler, D. E., eds), pp. 164-173, John Wiley & Sons, New York. Jiiger, J., KShler, E., Tucker, P., Sander, U., Housley-Markovic, Z., Fotheringham, I., Edwards, M., Hunter, M., Kirschner, K. & Jansonius, J. N. (1989). Crystallization and Preliminary X-ray Studies of an Aspartate Aminotransferase Mutant from Eseherichia coll. J. Mol. Biol. 209, 499-501. J~iger, J., Moser, M., Pauptit, R., Sauder, U., Tucker, P., K6hler, E., Seville, M., Christen, P., Kirschner, K. & Jansonius, J. (1990). X-ray Studies of Mutants and Inhibitor Complexes of Aspartate Aminotransferase from E. coll. Experientia, 46, A35. Jansonius, J. N. & Vincent, M. G. (1987). Structural Basis for Catalysisby Aspartate Aminotransferase. In Biological Macromolecules d: Assemblies (Jurnak, F. A. & McPherson, A., eds), vol. 3, Active Sites of Enzymes, pp. 187-285, John Wiley & Sons, New York. Jansonius, J. N., Vincent, M. G., McPhalen, C. A. & Picot, D. (1987). Spatial Aspects of Catalysis by Mitochondrial Aspartate Aminotransferase. In Biochemistry of Vitamin B 6 (Korpela, T. & Christen, P., eds), pp. 89-98, Birkh~iuser, Basel. Kamitori, S., Hirotsu, K., Higuchi, T., Kondo, K., Inoue, K., Kuramitsu, S., Kagamiyama, H., Higuchi, Y., Yasuoka, N., Kusunoki, M. & Matsuura, Y. (1987). Overproduction and Preliminary X-ray Characterization of Aspartate Aminotransfcrase from Escherichia coli. J. Biochem. (Tokyo), 101,813-816. Kamitori, S., Hirotsu, K., Higucbi, T., Kondo, K., ]noue, K., Katsura, S., Kuramitsu, S., Kagamiyama, H., Higuchi, Y., Yasuoka, N., Kusunoki, M. & Matsuura, Y. (1988). Three-Dimensional Structure of Aspartate
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Aminotransferase from Escherichia coli at 2-8A Resolution. J. Biochem. (Tokyo), 104, 317-318. Kitsch, J. F., Eiehele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H. & Christen, P. (1984). Mechanism of Action of Aspartate Aminotransferase Proposed on the Basis of its Spa:ial Structure. J. Mol. Biol. 174, 497-525. Lowe, C. R. & Pearson, J. C. (1984). Affinity Chromatography on Immobilized Dyes. Methods Enzymol. 104, 97-113. Matthews, B. W. (1968). Solvent Content of Protein Crystals. J. Mol. Biol. 33,491-497. Mehta, P. K., Hale, T. I. & Christen, P. (1989). Evolutionary Relationships Among Aminotransferases: Tyrosine Aminotransferase, Histidinol Phosphate Aminotransferase, and Aspartate Aminotransferase are Homologous Proteins. Eur. J. Biochem. 186, 249-253. Picot, /). (1987). Conformational changes of Mitochondrial Aspartate Aminotransferase during
Catalysis: An X-ray Crystallographic and Microspectrophotometrie Study. Ph.D thesis, University of Basel. Severin, E. S., Gulyaev, N. N., Khurs, E. N. & Khomutov, R. M. (1969). The Synthesis and Properties of Phosphopyridoxyl Amino Acids. Biochem. Biophys. Res. Commun. 35, 318--323. Smith, D. L., Ringe, D., Finlayson, W. L. & Kirsch, J. F. (1986}. Preliminary X-ray Data for Aspartate Aminotransferase from Escherichia coll. J. Mol. Biol. 191,301-302. Smith, D. L., Almo, S. C., Toney, M. D. & Ringe, D. (1989). 2"8A Resolution Crystal Structure of an Active Site Mutant of Aspartate Aminotransferase from Escherichia coli. Biochemistry, 28, 8161-8167. Thaller, C., Weaver, L. H., Eichele, G., Wilson, E., Karlsson, R. & Jansonius, J. N. (1981}. Repeated Seeding Technique for Growing Large Single Crystals of Proteins. J. Mol. Biol. 147, 465-469.
Edited by R. Huber
Note added in proof. The structure has been solved by molecular replacement. The space group is P3121. Rings of 12 dimers surround the 3t-axis forming solvent channels of 130 A diameter.
