J. Mol. Biol. (1990) 215, 211-213
Crystallographic Studies of 3-KetoacylCoA Thiolase from Yeast Saccharomyces cerevisiae J. Ph. Zeelen, R. K. Wierenga~f European Molecular Biology Laboratory Meyerhofstrafle 1, 6900 Heidelberg, F.R.G.
R. Erdmann and W.-H. Kunau R u h r Universitdt Bochum, Medizinische Fakultdt Universitdtsstrafle 150, 4630 Bochum, F.R.G.
(Received 21 May 1990; accepted 24 May 1990) Good diffracting crystals of 3-ketoacylCoA thiolase (EC 2.3.1.16) from yeast Saccharomyces cerevisiae have been obtained. The crystals diffract to at least 2-4 A. The space group of these crystals is P212121, with cell dimensions a - - 7 1 . 8 A, b = 93"8 A and c--119.9 A. There is one dimer per asymmetric unit.
Thiolases are ubiquitous enzymes involved in various different metabolic pathways. Several types of thiolases have been observed in eukaryotic and prokaryotic cells with differences in substrate specificity and intracellular localization. According to the former property" one can distinguish (1) thiolase with broad chain-length specificity (thiolase I or 3ketoacylCoA thiolase) associated with the fl-oxidation cycle for the degradation of fatty acids (Kunau et al., 1988), and (2) thiolase involved in the metabolism of acetoacetylCoA (thiolase II or acetoacetylCoA thiolase) associated with ketone-body metabolism (Middleton, 1973) and steroid biosynthesis (Davis et al., 1987a). With regard to intracellular localization, thiolases have been found in the cytoplasm, in mitochondria and in peroxisomes. Mitochondriai 3-ketoacylCoA thiolases from mammalian liver (Arakawa et al., 1987) have been extensively studied over a long period of time, while those of peroxisomes have recently been described from a few sources only (rat liver, Hijikata et al., 1987, 1989; cucumber seeds, Behrends et al., 1988; various yeasts, Kunau et al., 1988). All of these thiolases were found to consist of polypeptides of very similar size. Nevertheless, there are interesting differences between mitochondrial and peroxisomal 3-ketoacylCoA thiolases. While the former are tetrameric proteins the latter have been reported to contain only two identical subunits (Kunau et al.,
t Author to whom all correspondence should be addressed. 0022-2836/90/180211-03 $03.00/0
1988). The import mechanism that controls the cellular localization of the thiolases is not yet understood. Interestingly, the peroxisomal thiolase of rat liver is synthesized as a larger percursor (Hijikata et al., 1987), which is unusual for peroxisomal proteins, whereas the mitochondrial thiolase has been reported to lack a presequence (Amaya et al., 1988), unlike most other mitochondrial proteins. Sequences of several thiolases have been determined. These include mitochondrial 3-ketoacylCoA thiolase from rat liver (Arakawa et al., 1987), peroxisomal 3-ketoacylCoA thiolase from human liver (Bout et al., 1988) and from rat liver (Hijikata et al., 1987), as well as those from the yeasts Candida tropicalis (U. Stank & W.-H. Kunau, personal communication) and Saccharomyces cerevisiae (B. van Geldern & W.-H. Kunau, personal communication). The primary structures of acetoacetylCoA thiolase from rat liver mitochondria (Fukao et al., 1989), from the yeast S. uvarum (Dequin et al., 1988) as well as from the bacterium Zoogloea ramigera (Peoples et al., 1987), have been reported. Sequence comparison shows that all thiolases are a family of rather conserved proteins of approximately 420 residues, that show no similarity to other proteins. The cysteine residue, which is predicted to be part of the active centre of bacterial acetoacetylCoA thiolase (Davis et al., 1987b), is algo present in corresponding positions of all other known thiolase sequences. This conserved cysteine is anticipated from an acetyl-S-enzyme intermediate in the catalytic reaction (Davis et al., 1987b). 211
© I99OAcademic Press Limited
212
J. Ph. Zeelen et al.
Recently, thiolases have aroused medical interest because deficiencies of different thiolases are reported to account for inherited diseases (Schram et al., 1987; Nagasawa et al., 1989). In the crystallographic investigations described here, the 3-ketoacylCoA thiolase from the peroxisomes of the yeast S. cerevisiae is being studied. This enzyme catalyses the thiolytie cleavage of 3-ketoacylCoA: 3-ketoacylCoA + CoA -* AcetylCoA + AcylCoA Therefore, this enzyme must have a rather extensive active site with a binding pocket for 3-ketoacylCoA and CoA. Structures of some acetylCoA or acylCoA-binding enzymes are known, such as from porcine heart mitochondrial citrate synthase (Wiegand et al., 1984), porcine heart mitochondrial L-3-hydroxyacylCoA dehydrogenase (Birktoft et al., 1987) and porcine liver mitochondrial mediumchain acylCoA dehydrogenase (Kim et al., 1988). However, thiolases so far have not been studied crystallographically. Our crystallization attempts were initiated once a suitable overexpression system was obtained. For this reason the thiolase gene of the peroxisomal S. cerevisiae thiolase was introduced into the multicopy vector YEp352. Transformation of the protease-deficient yeast strain ABYS1 with this vector led to a 50-fold overexpression of thiolase. This provided the opportunity to implement a purification protocol, by which approximately 10 mg per batch can be purified routinely (B. van Geldern & W.-H. Kunau, personal communication). The successful crystallization experiments have been carried out using a dialysis technique (Phillips, 1985). An enzyme solution consisting of 10 mg of thiolase/ml in 25 mM-Tris-HCl (pH 7"4), 200 mmpotassium phosphate, 1 mM-EDTA, 1 mM-dithiothreitol (DTTt), 1 mM-sodium azide, was dialysed against a phosphate-free buffer (25 mM3-(N-morpholino)-propane sulphonic acid (pH 6"5), 1 mm-EDTA, 1 mM-DTT, 1 mM-sodium azide), at room temperature. Crystals grew in a few days. These dialysis experiments were done in small glass capillaries, as shown in Figure 1. As the dialysis membrane, a crosslinked polyacrylamide gel was used that can be made in the following way. As soon as the polymerization in a solution containing 15~o (w/v) acrylamide, 0"5% (w/v) bisaerylamide, 200mM-phosphate (pI-I 7.4), is initiated, small quantities of the polymerizing solution are injected into the end of the capillaries. The injection volume determines the-height Of the gel column. Before use, the gels are washed by filling the capillaries with buffer (25 mM-Tris-HCI (pH 7"4), 200 mM-potassium phosphate, 1 mM-EDTA, 1 mM-DTT, 1 mM-sodium azide), and placing them for two days in a reservoir with the same buffer. The best results are obtained with a 4 mm gel column and 15% acrylamide, 0"5% bisacrylamide. t Abbreviation used: DTT, dithiothreitol.
--I
! mffl
I--
i i
.,.l"llA "/A
Protein solution Polyocrylomide gel Reservoir s o l u t i o n Gloss fibres to support the C o p i l l o r y
Figure 1. Crystallization in glass capillaries, using the polyacrylamide gel dialysis technique.
The crystals are of reasonable size, such as 0"3 mm x 0"3 mm x 0"15 mm. The crystals diffract to at least 2-4 A (1 A = 0"1 nm), but after one day of exposure the diffracting power is considerably reduced. Precession pictures have been taken from the three major zones as well as from some first upper levels. The X-ray diffraction pattern has the symmetry and extinctions of space group P21212 I. The cell dimensions are a --- 71"8 A, b = 93"8 A and c = 119"9 A. There is probably one dimer per asymmetric unit, yielding a Vm of 2"3 _h_a/Da (Matthews, 1968). It will be attempted to reduce the radiation damage of the crystals by cooling methods. Currently these crystals are also used for heavyatom screening experiments. Once suitable heavyatom derivatives have been found then the structure can be solved via the multiple isomorphous replacement method. The atomic structure of thiolase might clarify some of the aspects of the peroxisomal import mechanism, it will also give an insight into the catalytic mechanism of this enzyme. We are grateful to Barbara vail Geldern for the modified purification protocol and Ursula Dorpmund for the purification of the thio[ase. We thank Dr Werner Kiihlbrandt and Karoline D6rr for discussions about the crystallization protocol and Dr Paul Tucker for measuring the cell dimensions on the FAST area detector. References
Amaya, Y., Arakawa, H., Takiguchi, M., Ebina, Y., Yokota, S. & Mori, M. (1988). A Noncleavable Signal for Mitochondriai Import of 3-Oxoacyl-CoAThiolase. J. Biol. Chem. 263, 14463-14470. Arakawa, H., Takiguchi, M., Amaya, Y., Nagata, S., Hayashi, H. & Mori, M. (1987). cDNA-derived Amino Acid Sequence of Rat Mitochondrial 3-oxoacyI-CoA Thiolase with no Transient Presequence: Structural Relationship with Peroxisomal Isozyme. E M B O J. 5, 1361-1366.
Communications
Behrends, W., Thieringer, R., Engeland, K., Kunau, W.-H. & Kindl, H. (1988). The Glyoxysomal fl-Oxidation System in Cucumber Seedlings: Identification of Enzymes Required for the Degradation of Unsaturated Fatty Acids. Arch. Biochem. Biophys. 263, 170-177. Birktoft, J. J., Holden, H. M., Hamlin, R., Xuong, N. H. & Banaszak, L. J. (1987). Structure of L-3-HydroxyacyI-Coenzyme A Dehydrogenase: Preliminary Chain Tracing at 2"8 A Resolution. Proc. Nat. Acad. Sci., U.S.A. 84, 8262-8266. Bout, A., Teunissen, Y., Hashimoto, T., Benne, R. & Tager, J. M. (1988). Nucleotide Sequence of Human Peroxisomal 3-Oxoacyl-CoA Thiolase. Nucl. Acids Res. 16, 10369. Davis, J. T., Moore, R. N., Imperiali, B., Pratt, A. J., Kobayashi, K., Masamune, S., Sinskey, A. J., Walsh, C. T., Fukui, T. & Tomita, K. (1987a). J. Biol. Chem. 262, 82-89. Davis, J. T., Chen, H.-H., Moore, R. N., Nishitani, Y., Masamune, S., Sinskey, A. J. & Walsh, C. T. (1987b). J. Biol. Chem. 262, 90-96. Dequin, S., Gloeckler, R., Herbert, C.J. & Boutelet, F. (1988). Curt. Genet. 13, 471-478. ' Fukao, T., Kamijo, K., Osumi, T., Fujiki, Y., Yamaguchi, S., Orii, T. & Hashimoto, T. (1989). J. Biochem. 106, 197-204. Hijikata, M., Ishii, N., Kagamiyama, H., Osumi, T. & Hashimoto, T. (1987). J. Biol. Chem. 262, 8151-8158.
213
Hijikata, M., Wen, J.-K., Osumi, T. & Hashimoto, T. (1989). J. Biol. Chem. 265, 4600-4606. Kim, J.-J. P. & Wu, J. (1988). Proc. Nat. Acad. Sci., U.S.A. 85, 6677-6681. Kunau, W.-H., Biihne, S., de la Garza, M., Kionka, C., Mateblowski, M., Schultz-Borchard, U. & Thieringer, R. (1988). Biochem. Soc. Trans. 16, 418-420. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Middleton, B. (1973). Biochem. J. 132, 717-730. Nagasawa, H., Yamaguchi, S., Orii, T., Schutgens, R. B. H., Sweetman, L. & Hashimoto, T. (1989). Pediatr. Res. 26, 145-149. Peoples, O. P., Masamune, S., Walsh, C. T. & Sinskey, A. T. (1987). J. Biol. Chem. 262, 97-102. Phillips, G. N. Jr (1985). Methods Enzymol. 114, 128-131. Schram, A. W., Goldfischer, S., van Roermund, C. W. T., Brouwer-Kelder, E. M., Collins, J., Hashimoto, T., Heymans, H. S. A., van den Bosch, H., Sehutgens, R. B. H., Tager, J. M. & Wanders, R. J. A. (1987). Human Peroxisomal 3-OxacyI-Coenzyme A Thiolase Deficiency. Proc. Nat. Acad. Sci., U.S.A. 84, 2494-2496. Wiegand, G., Remington, S., Deisenhofer, J. & Huber, R. (1984). Crystal Structure Analysis and Molecular Model of a Complex of Citrate Synthase with Oxaloacetate and S-Acetyonyl-Coenzyme A. J. Mol. Biol. 174, 205-219.
Edited by A. Klug
Crystallographic Studies of 3-KetoacylCoA Thiolase from Yeast Saccharomyces cerevisiae J. Ph. Zeelen, R. K. Wierenga~f European Molecular Biology Laboratory Meyerhofstrafle 1, 6900 Heidelberg, F.R.G.
R. Erdmann and W.-H. Kunau R u h r Universitdt Bochum, Medizinische Fakultdt Universitdtsstrafle 150, 4630 Bochum, F.R.G.
(Received 21 May 1990; accepted 24 May 1990) Good diffracting crystals of 3-ketoacylCoA thiolase (EC 2.3.1.16) from yeast Saccharomyces cerevisiae have been obtained. The crystals diffract to at least 2-4 A. The space group of these crystals is P212121, with cell dimensions a - - 7 1 . 8 A, b = 93"8 A and c--119.9 A. There is one dimer per asymmetric unit.
Thiolases are ubiquitous enzymes involved in various different metabolic pathways. Several types of thiolases have been observed in eukaryotic and prokaryotic cells with differences in substrate specificity and intracellular localization. According to the former property" one can distinguish (1) thiolase with broad chain-length specificity (thiolase I or 3ketoacylCoA thiolase) associated with the fl-oxidation cycle for the degradation of fatty acids (Kunau et al., 1988), and (2) thiolase involved in the metabolism of acetoacetylCoA (thiolase II or acetoacetylCoA thiolase) associated with ketone-body metabolism (Middleton, 1973) and steroid biosynthesis (Davis et al., 1987a). With regard to intracellular localization, thiolases have been found in the cytoplasm, in mitochondria and in peroxisomes. Mitochondriai 3-ketoacylCoA thiolases from mammalian liver (Arakawa et al., 1987) have been extensively studied over a long period of time, while those of peroxisomes have recently been described from a few sources only (rat liver, Hijikata et al., 1987, 1989; cucumber seeds, Behrends et al., 1988; various yeasts, Kunau et al., 1988). All of these thiolases were found to consist of polypeptides of very similar size. Nevertheless, there are interesting differences between mitochondrial and peroxisomal 3-ketoacylCoA thiolases. While the former are tetrameric proteins the latter have been reported to contain only two identical subunits (Kunau et al.,
t Author to whom all correspondence should be addressed. 0022-2836/90/180211-03 $03.00/0
1988). The import mechanism that controls the cellular localization of the thiolases is not yet understood. Interestingly, the peroxisomal thiolase of rat liver is synthesized as a larger percursor (Hijikata et al., 1987), which is unusual for peroxisomal proteins, whereas the mitochondrial thiolase has been reported to lack a presequence (Amaya et al., 1988), unlike most other mitochondrial proteins. Sequences of several thiolases have been determined. These include mitochondrial 3-ketoacylCoA thiolase from rat liver (Arakawa et al., 1987), peroxisomal 3-ketoacylCoA thiolase from human liver (Bout et al., 1988) and from rat liver (Hijikata et al., 1987), as well as those from the yeasts Candida tropicalis (U. Stank & W.-H. Kunau, personal communication) and Saccharomyces cerevisiae (B. van Geldern & W.-H. Kunau, personal communication). The primary structures of acetoacetylCoA thiolase from rat liver mitochondria (Fukao et al., 1989), from the yeast S. uvarum (Dequin et al., 1988) as well as from the bacterium Zoogloea ramigera (Peoples et al., 1987), have been reported. Sequence comparison shows that all thiolases are a family of rather conserved proteins of approximately 420 residues, that show no similarity to other proteins. The cysteine residue, which is predicted to be part of the active centre of bacterial acetoacetylCoA thiolase (Davis et al., 1987b), is algo present in corresponding positions of all other known thiolase sequences. This conserved cysteine is anticipated from an acetyl-S-enzyme intermediate in the catalytic reaction (Davis et al., 1987b). 211
© I99OAcademic Press Limited
212
J. Ph. Zeelen et al.
Recently, thiolases have aroused medical interest because deficiencies of different thiolases are reported to account for inherited diseases (Schram et al., 1987; Nagasawa et al., 1989). In the crystallographic investigations described here, the 3-ketoacylCoA thiolase from the peroxisomes of the yeast S. cerevisiae is being studied. This enzyme catalyses the thiolytie cleavage of 3-ketoacylCoA: 3-ketoacylCoA + CoA -* AcetylCoA + AcylCoA Therefore, this enzyme must have a rather extensive active site with a binding pocket for 3-ketoacylCoA and CoA. Structures of some acetylCoA or acylCoA-binding enzymes are known, such as from porcine heart mitochondrial citrate synthase (Wiegand et al., 1984), porcine heart mitochondrial L-3-hydroxyacylCoA dehydrogenase (Birktoft et al., 1987) and porcine liver mitochondrial mediumchain acylCoA dehydrogenase (Kim et al., 1988). However, thiolases so far have not been studied crystallographically. Our crystallization attempts were initiated once a suitable overexpression system was obtained. For this reason the thiolase gene of the peroxisomal S. cerevisiae thiolase was introduced into the multicopy vector YEp352. Transformation of the protease-deficient yeast strain ABYS1 with this vector led to a 50-fold overexpression of thiolase. This provided the opportunity to implement a purification protocol, by which approximately 10 mg per batch can be purified routinely (B. van Geldern & W.-H. Kunau, personal communication). The successful crystallization experiments have been carried out using a dialysis technique (Phillips, 1985). An enzyme solution consisting of 10 mg of thiolase/ml in 25 mM-Tris-HCl (pH 7"4), 200 mmpotassium phosphate, 1 mM-EDTA, 1 mM-dithiothreitol (DTTt), 1 mM-sodium azide, was dialysed against a phosphate-free buffer (25 mM3-(N-morpholino)-propane sulphonic acid (pH 6"5), 1 mm-EDTA, 1 mM-DTT, 1 mM-sodium azide), at room temperature. Crystals grew in a few days. These dialysis experiments were done in small glass capillaries, as shown in Figure 1. As the dialysis membrane, a crosslinked polyacrylamide gel was used that can be made in the following way. As soon as the polymerization in a solution containing 15~o (w/v) acrylamide, 0"5% (w/v) bisaerylamide, 200mM-phosphate (pI-I 7.4), is initiated, small quantities of the polymerizing solution are injected into the end of the capillaries. The injection volume determines the-height Of the gel column. Before use, the gels are washed by filling the capillaries with buffer (25 mM-Tris-HCI (pH 7"4), 200 mM-potassium phosphate, 1 mM-EDTA, 1 mM-DTT, 1 mM-sodium azide), and placing them for two days in a reservoir with the same buffer. The best results are obtained with a 4 mm gel column and 15% acrylamide, 0"5% bisacrylamide. t Abbreviation used: DTT, dithiothreitol.
--I
! mffl
I--
i i
.,.l"llA "/A
Protein solution Polyocrylomide gel Reservoir s o l u t i o n Gloss fibres to support the C o p i l l o r y
Figure 1. Crystallization in glass capillaries, using the polyacrylamide gel dialysis technique.
The crystals are of reasonable size, such as 0"3 mm x 0"3 mm x 0"15 mm. The crystals diffract to at least 2-4 A (1 A = 0"1 nm), but after one day of exposure the diffracting power is considerably reduced. Precession pictures have been taken from the three major zones as well as from some first upper levels. The X-ray diffraction pattern has the symmetry and extinctions of space group P21212 I. The cell dimensions are a --- 71"8 A, b = 93"8 A and c = 119"9 A. There is probably one dimer per asymmetric unit, yielding a Vm of 2"3 _h_a/Da (Matthews, 1968). It will be attempted to reduce the radiation damage of the crystals by cooling methods. Currently these crystals are also used for heavyatom screening experiments. Once suitable heavyatom derivatives have been found then the structure can be solved via the multiple isomorphous replacement method. The atomic structure of thiolase might clarify some of the aspects of the peroxisomal import mechanism, it will also give an insight into the catalytic mechanism of this enzyme. We are grateful to Barbara vail Geldern for the modified purification protocol and Ursula Dorpmund for the purification of the thio[ase. We thank Dr Werner Kiihlbrandt and Karoline D6rr for discussions about the crystallization protocol and Dr Paul Tucker for measuring the cell dimensions on the FAST area detector. References
Amaya, Y., Arakawa, H., Takiguchi, M., Ebina, Y., Yokota, S. & Mori, M. (1988). A Noncleavable Signal for Mitochondriai Import of 3-Oxoacyl-CoAThiolase. J. Biol. Chem. 263, 14463-14470. Arakawa, H., Takiguchi, M., Amaya, Y., Nagata, S., Hayashi, H. & Mori, M. (1987). cDNA-derived Amino Acid Sequence of Rat Mitochondrial 3-oxoacyI-CoA Thiolase with no Transient Presequence: Structural Relationship with Peroxisomal Isozyme. E M B O J. 5, 1361-1366.
Communications
Behrends, W., Thieringer, R., Engeland, K., Kunau, W.-H. & Kindl, H. (1988). The Glyoxysomal fl-Oxidation System in Cucumber Seedlings: Identification of Enzymes Required for the Degradation of Unsaturated Fatty Acids. Arch. Biochem. Biophys. 263, 170-177. Birktoft, J. J., Holden, H. M., Hamlin, R., Xuong, N. H. & Banaszak, L. J. (1987). Structure of L-3-HydroxyacyI-Coenzyme A Dehydrogenase: Preliminary Chain Tracing at 2"8 A Resolution. Proc. Nat. Acad. Sci., U.S.A. 84, 8262-8266. Bout, A., Teunissen, Y., Hashimoto, T., Benne, R. & Tager, J. M. (1988). Nucleotide Sequence of Human Peroxisomal 3-Oxoacyl-CoA Thiolase. Nucl. Acids Res. 16, 10369. Davis, J. T., Moore, R. N., Imperiali, B., Pratt, A. J., Kobayashi, K., Masamune, S., Sinskey, A. J., Walsh, C. T., Fukui, T. & Tomita, K. (1987a). J. Biol. Chem. 262, 82-89. Davis, J. T., Chen, H.-H., Moore, R. N., Nishitani, Y., Masamune, S., Sinskey, A. J. & Walsh, C. T. (1987b). J. Biol. Chem. 262, 90-96. Dequin, S., Gloeckler, R., Herbert, C.J. & Boutelet, F. (1988). Curt. Genet. 13, 471-478. ' Fukao, T., Kamijo, K., Osumi, T., Fujiki, Y., Yamaguchi, S., Orii, T. & Hashimoto, T. (1989). J. Biochem. 106, 197-204. Hijikata, M., Ishii, N., Kagamiyama, H., Osumi, T. & Hashimoto, T. (1987). J. Biol. Chem. 262, 8151-8158.
213
Hijikata, M., Wen, J.-K., Osumi, T. & Hashimoto, T. (1989). J. Biol. Chem. 265, 4600-4606. Kim, J.-J. P. & Wu, J. (1988). Proc. Nat. Acad. Sci., U.S.A. 85, 6677-6681. Kunau, W.-H., Biihne, S., de la Garza, M., Kionka, C., Mateblowski, M., Schultz-Borchard, U. & Thieringer, R. (1988). Biochem. Soc. Trans. 16, 418-420. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Middleton, B. (1973). Biochem. J. 132, 717-730. Nagasawa, H., Yamaguchi, S., Orii, T., Schutgens, R. B. H., Sweetman, L. & Hashimoto, T. (1989). Pediatr. Res. 26, 145-149. Peoples, O. P., Masamune, S., Walsh, C. T. & Sinskey, A. T. (1987). J. Biol. Chem. 262, 97-102. Phillips, G. N. Jr (1985). Methods Enzymol. 114, 128-131. Schram, A. W., Goldfischer, S., van Roermund, C. W. T., Brouwer-Kelder, E. M., Collins, J., Hashimoto, T., Heymans, H. S. A., van den Bosch, H., Sehutgens, R. B. H., Tager, J. M. & Wanders, R. J. A. (1987). Human Peroxisomal 3-OxacyI-Coenzyme A Thiolase Deficiency. Proc. Nat. Acad. Sci., U.S.A. 84, 2494-2496. Wiegand, G., Remington, S., Deisenhofer, J. & Huber, R. (1984). Crystal Structure Analysis and Molecular Model of a Complex of Citrate Synthase with Oxaloacetate and S-Acetyonyl-Coenzyme A. J. Mol. Biol. 174, 205-219.
Edited by A. Klug
