J. Mol. Riol. (1991) 221, 1075-1077
Crystallization
and Crystal Packing of Proteus mirabilis PR Catalase
HCl&ne-Marie Jouve’t, Patrice Gouet’, Nassira Boudjada’, Georges Buisson’ Richard Kahn3 and Emile Duee’ ‘Laboratoire ‘Laboratoire 3LURE
de Biologic Structurale, CEA et URA 1333 CNRS, BP 85X, 38041 Grenoble Gedex, France. de Cristallographie,
CNRS,
BP 166X,
DBMSjDSF
38042 Grenoble Cedex, France
(CNRS & Universite’ Paris-Sud), 91405 Orsay Cedex et LPCS Universite’ Val de Marne, avenue ge’ne’rale de Gaulle, 94000 Creteil, France (Received 22 April
1991; accepted 5 July
Paris
1991)
The tetrameric catalase from Proteus mirabilis PR (EC 1.11.1.6), known to bind NADPH, has been crystallized by the hanging-drop method in a form apparently depleted in dinucleotide. The crystals belong to the hexagonal space group P6,22 with a = b = 111.7 8, c = 248.8 8. There is one subunit in the asymmetric unit. Data were collected to 2.9 i! at the L.C.R.E. (Orsay) synchrotron radiation facility. The tetramers have been located in the crystal, centered on the site (l/2,0,0) with 222 symmetry.
Keywords: catalase;
NADPH;
crystallization;
Catalase (EC 1.11.1.6), which is present in almost’ all aerobic organisms, is known for its ability to decompose hydrogen peroxide to molecular oxygen and water (Deisseroth & Dounce, 1970). Its exact physiological role, however, still remains unclear. Catalases from certain species have been shown to contain tightly bound NADPH (Kirkman & Gaetani. 1984). Tt is thought that this NADPH acts t.o protect the enzyme against inactivation by its substrate, although ?;ADPH does not appear to be present in all catalases (Kirkman et al., 1987; Beaumont et al.. 1990). Three tet’rameric catalase structures, from I’enicillium vitale (Vainshtein et al., 1986), from beef liver (Murthy et al., 1981) and from Micrococcus lysodeikticu,s (Yusifov et al., 1989) are now known at high resolution. The electron density maps of catalases from beef liver and M. lysodeikticus confirm the presence of one NADP molecule for each P. witale catalase differs from monomer. However, the other two in having a flavodoxin-like domain (a/j3 structure) on the carboxyl end of the polypeptide chain. Previous work with catalase from Proteus mirahilis PR, a mutant with high resistance to H,O,, showed that two different forms of this enzyme, named A and B, could be separated by anion exchange chromatography and further purified to homogeneity (Jouve et al., 1983). The cata-
t Author
t,o whom all correspondence
should be sent.
X-ray
diffraction;
crystal-packing
lase B contained tightly bound NADPH, while the A form was apparently depleted in dinucleotide (Jouve et al., 1989). The crystallization, preliminary X-ray diffraction analysis, and crystal packing of the A form of P. mirabilis PR catalase are reported here. P. mirabilis PR catalase A was purified using the method previously described (Jouve et al., 1983). was The protein stored at -20°C in 100 mM-Tris . HCl (pH 7.5) containing glycerol in the proportion of 10 : 1 (v/v). Crystallization was performed using the vapor diffusion method (hanging drop) at a temperature of 4 t’o 5”C, with ammonium sulfate as precipitating agent. The experimental conditions were as follows: 3 ~1 of the catalase solution at an initial concentration of 54 mg ml-’ in 100 mM-Tris.HCl (pH 7.5). 5% (v/v) glycerol, was mixed in a droplet’ with an equal containing 2 Mvolume of reservoir solution ammonium sulfate, 50 mM-KCl, 100 mM-Tris* HCl buffered within the pH range 6.0 to 8.0. Brown-colored hexagonal bipyramidal crystals grew within a week. The largest crystals of a size about 1.0 mm x 0.5 mm x @5 mm were obtained between pH 6.5 and 7.5. The crystals were very temperature-sensitive and dissolved at temperatures above 10°C. Crystals were transferred to and kept in 3.7 M-ammonium sulfate, 50 mM-KCl, 100 mM-Tris. HCl (pH 7.5). The addition of NADPH to the crystal growth prevented the crystallization of the mixture 1075
0 1991 Academic Press Limited
1076
H.-M. Jouve et al.
enzyme. However, the crystals of catalase A could be soaked in 10 mM-NADPH without any apparent, changes in the crystal parameters and symmetry. Crystals of catalase A, soaked in NADPH and then carefully washed four times with dinucleotide-free sulfate, could be dissolved in ammonium 100 mM-Tris buffer (pH 7.5). The enzyme) thus obtained, had the same biochemical characteristics as the B form: behavior in anion exchange chromatography and ability to reverse the complex II formation (Jouve et al., 1989). This suggested that NADPH was bound to the enzyme molecule in the crystal. CuKcl radiation was used with a conventional X-ray generator or with a rotating anode equipped with double mirrors to collimate the beam and separate diffraction spots corresponding to large cell parameters. The crystals diffract to 2.4 A resolution (1 A = 0.1 nm) and are stable in the X-ray beam for about 100 hours. X-ray precession photographs showed systematic reflection conditions on OOL, showed an hexagonal I, = 3n. HKO and HKI symmetry. and HOL, HlL, H2L showed two perpendicular mirror planes giving for the space groups either P6222 or its enantiomorph P6,22. The hexagonal unit cell parameters are n = b = 111.7 A. c = 249.8 A. A crystal density of 1.25 was found when measured on a gradient of carbon tetrachloride in water-saturated toluene (Matthews, 1968, 1985). From the volume of the unit cell, the number of tetrameric molecules per unit cell was calculated to be three and the solvent fraction 63%. Other possibilities are outside the range of reasonable values.
In space group P622. the number of eyuivalent positions is 12. consequently, the asymmetric unit contains one monomer. as for the M. lysodeikticus enzyme (Yusifov ef al., 1989). The catalasr of P. mirabiZia PR exhibits a true molecular 222 symmetry, whereas the symmetry is only nearIS 2-fold for the tetrameric catalase of beef liver and P. vitale (Melik-Adamyan et al., 1986). An initial data collection was ca,rried out at the L.C.R.E. synchrotron radiation facility on the 21) MARK2 electronic detector (Kahn et al.. 1986). Two crystals were used. one with c* along t,hr rotat*ion axis from 0” t)o 60” in phi and the &her with o* along the rotation axis beginning with (A* perpendicular to the X-ray beam (O” to 12” in phi). A tot,al of 48,842 diffract’ion intensities were collected to 2.9 a during the first c*ollection. giving 17.401 unique reflections after using the program PRO(‘OR of MADNES (Kabsch. 1988) in the space group P6222. This represent,s nearly 90(!,, of the complete data with an agreement factor I&,,,, = 4%(),, on fquivalent reflections and intensities. Since the unit, cell contains t#hree t,etrameric~ molrcules wit,h molecular spmmetry 222. t,he center of the tetramer must be on one of the positions of the P6*22 or (P6,22) cell with a 222 site1 symmetry. Moreover, the molecular axes P, &. R (Murbhy CJ~ trl.. 1981) must he aligned with the t,hree crystallo.graphic dyad axes intersecting atI this 222 sit)e. In each of the enantiomorphic spaccb groups, two different positions arc possible. namelv (0. 0. 0) or (l/2, 0. 0) with six different possible or&iations at each posit’ion. The R-factor. between observed and ~&uIated
Figure 1. Projection down the e2 axes. of the crystal structure of P. mirabiZi,s PR catalase The molecaular cent,rrs are positioned on the crystallographic sites with 222 symmetry at co-ordinates (l/‘L. 0. 0). (l/Z. l/2. 10). (0. 112, g/3). FIW solvent forms cylinders around the 6, axes with a diameter of 45 .+%shown in t,he Figure and channels orthogonal to the 6* axes (not shown).
Communications structure factors for data in the range 8 to 4 A, was computed with the program CORELS (Sussman, 1985) for each possible solution (co-ordinates of the molecular center and orientation), using as a model, the beef liver catalase (Fita & Rossmann, 1985) from the Protein Data Bank. In space group P6,22, R-factors for all possible solutions were approximately 56O/,. In space group P6222, centering the tetramer at (l/2, 0.0) with its P axis parallel to the 6-fold crystallographic axis and its & axis along a, gave an R-factor of 44%, while the other orientations and centerings gave an R-factor near 56O&. Using the beef liver catalase co-ordinates as a model for the P. mirabilis PR catalase, the packing of the molecules in the crystal is shown in Figure 1. Refinement and determination of the primary struct#ure are under
way.
References Beaumont, F.. Jouve, H. M., Gagnon, ,J., Gaillard, J. & Pelmont. J. (1990). Purification and properties of a catalase from potato tubers (Solanum tuberosum). Hunt A%. 72, 19-26. Deisseroth. A. & Dounce. A. L. (1970). Catalase: physical and chemical properties, mechanism of catalysis, and physiological role. Physiol. Rev. 50, 319-375. Fita. T. & Rossman. M. G. (1985). The NADPH binding site on beef liver catalase. Proc. Na.t. Acad. SC%., 1:S.A. 82, 1604-1608. Jouve, H. M., Lasaunikre, C. & Pelmont? J. (1983). Properties of a catalase from a peroxide-resistant mutant of Proteus mirabilis. Canad. J. Riochem. Cull Bid. 61. 1219.-1227. ,Jouve. H. M., Beaumont, F., Lkger, I.. Foray, J. & Pelmont. .J. (1989). Tightly bound KADPH in Proteus mirabilis catalase. Biochem. Cell Biol. 67: 27 I-277. Kattsh. EV. (1988). Evaluation of single crystal X-ray difli-action data from a position sensitive detector. J. Appl. C’rystallogr. 21. 916-934.
1077
Kahn, R., Fourme, R. & Bosshard, R. (1986). An areadetector diffractometer for the collection of high resolution and multiwavelength anomalous diffraction data in macromolecular crystallography. Nucl. Znstr. Meth. A246, 596-603. Kirkman, H. xi. & Gaetani, G. F. (1984). Catalase: a tetrameric enzyme with four tightly bound molecules Proc. Nut. ilcad. Sci.. I:.S.A. 81, of KADPH. 4343-4347. Kirkman, H. N., Galiano, S. & Gaetani, G. F. (1987). The function of catalase-bound NADPH. ./. Kiol. Chem. 262. 660-666.
Matthews, B. W. (1968). Solvent content of protein crystals. d. Mol. Biol. 33. 491-497. Matthews, B. W. (1985). Determination of protein molecular weight, hydration, and packing from crystal density. In Methods in &zymology (Colowick. S. P. & Kaplan, N. 0.. eds), vol. 114. pp. 176-187. Academic Press. New York. Melik-Adamyan, W. R.. Barynin, V. V.. Vagin, A. A.. Borisov. V. V.. Vainshtein. B. K.. Fita. I.. Murthp. M. R. N. & Rossmann, M. G. (I 986). Comparison of beef liver and Penkillium vita/e catalasrs. J. Mol. Riol. 188. 63-72. Murthy. II. R. N;., Reid. T. ,J.. III, Sicignano. A., Tanaka. N. & Rossman, M. G. (1981). St’ruc*turc> of beef liver catalase. ,I. Mol. Biol. 152. 465-499. Sussman, ,J. L. (1985). Constrained-restrained leastsquares ((IORELS) refinement of proteins and nucleic (Colowick, S. P. & acids. In Methods in &zymology Kaplan. S. 0.: eds), vol. 11.5, pp. 271.-303. Academic, Press. &w York. Vainshtrin. B. K.. Melik-Adamyan. W. R.. Barynin. V. V., Vagin, A. A., Grebenko. .4. T.. Borisov. 3. V.. Bartels. K. S.. Fita. I. & Rossman. M. G. (1986). Three-dimensional of ratalase structure from Penicilliu,m vitale at 2.0 p\ resolut,ion. .I. ~Vol. Biol. 188. 49-61. Yusifov. E. F.. Grebenko, 9. I.. Barynin, V. V.. Murshudov. G. E;., Vagin. A. A.. Melik-Adamyan. V. R. & Vainshtein. B. K. (1989). Three-dimensional structure of ratalase from Micrococczts lysodeikticus at a resolution of 3.0 A. SOT:. Phys. Cry.stallogr. 34. 870-854.
Edited by R. Huber
Crystallization
and Crystal Packing of Proteus mirabilis PR Catalase
HCl&ne-Marie Jouve’t, Patrice Gouet’, Nassira Boudjada’, Georges Buisson’ Richard Kahn3 and Emile Duee’ ‘Laboratoire ‘Laboratoire 3LURE
de Biologic Structurale, CEA et URA 1333 CNRS, BP 85X, 38041 Grenoble Gedex, France. de Cristallographie,
CNRS,
BP 166X,
DBMSjDSF
38042 Grenoble Cedex, France
(CNRS & Universite’ Paris-Sud), 91405 Orsay Cedex et LPCS Universite’ Val de Marne, avenue ge’ne’rale de Gaulle, 94000 Creteil, France (Received 22 April
1991; accepted 5 July
Paris
1991)
The tetrameric catalase from Proteus mirabilis PR (EC 1.11.1.6), known to bind NADPH, has been crystallized by the hanging-drop method in a form apparently depleted in dinucleotide. The crystals belong to the hexagonal space group P6,22 with a = b = 111.7 8, c = 248.8 8. There is one subunit in the asymmetric unit. Data were collected to 2.9 i! at the L.C.R.E. (Orsay) synchrotron radiation facility. The tetramers have been located in the crystal, centered on the site (l/2,0,0) with 222 symmetry.
Keywords: catalase;
NADPH;
crystallization;
Catalase (EC 1.11.1.6), which is present in almost’ all aerobic organisms, is known for its ability to decompose hydrogen peroxide to molecular oxygen and water (Deisseroth & Dounce, 1970). Its exact physiological role, however, still remains unclear. Catalases from certain species have been shown to contain tightly bound NADPH (Kirkman & Gaetani. 1984). Tt is thought that this NADPH acts t.o protect the enzyme against inactivation by its substrate, although ?;ADPH does not appear to be present in all catalases (Kirkman et al., 1987; Beaumont et al.. 1990). Three tet’rameric catalase structures, from I’enicillium vitale (Vainshtein et al., 1986), from beef liver (Murthy et al., 1981) and from Micrococcus lysodeikticu,s (Yusifov et al., 1989) are now known at high resolution. The electron density maps of catalases from beef liver and M. lysodeikticus confirm the presence of one NADP molecule for each P. witale catalase differs from monomer. However, the other two in having a flavodoxin-like domain (a/j3 structure) on the carboxyl end of the polypeptide chain. Previous work with catalase from Proteus mirahilis PR, a mutant with high resistance to H,O,, showed that two different forms of this enzyme, named A and B, could be separated by anion exchange chromatography and further purified to homogeneity (Jouve et al., 1983). The cata-
t Author
t,o whom all correspondence
should be sent.
X-ray
diffraction;
crystal-packing
lase B contained tightly bound NADPH, while the A form was apparently depleted in dinucleotide (Jouve et al., 1989). The crystallization, preliminary X-ray diffraction analysis, and crystal packing of the A form of P. mirabilis PR catalase are reported here. P. mirabilis PR catalase A was purified using the method previously described (Jouve et al., 1983). was The protein stored at -20°C in 100 mM-Tris . HCl (pH 7.5) containing glycerol in the proportion of 10 : 1 (v/v). Crystallization was performed using the vapor diffusion method (hanging drop) at a temperature of 4 t’o 5”C, with ammonium sulfate as precipitating agent. The experimental conditions were as follows: 3 ~1 of the catalase solution at an initial concentration of 54 mg ml-’ in 100 mM-Tris.HCl (pH 7.5). 5% (v/v) glycerol, was mixed in a droplet’ with an equal containing 2 Mvolume of reservoir solution ammonium sulfate, 50 mM-KCl, 100 mM-Tris* HCl buffered within the pH range 6.0 to 8.0. Brown-colored hexagonal bipyramidal crystals grew within a week. The largest crystals of a size about 1.0 mm x 0.5 mm x @5 mm were obtained between pH 6.5 and 7.5. The crystals were very temperature-sensitive and dissolved at temperatures above 10°C. Crystals were transferred to and kept in 3.7 M-ammonium sulfate, 50 mM-KCl, 100 mM-Tris. HCl (pH 7.5). The addition of NADPH to the crystal growth prevented the crystallization of the mixture 1075
0 1991 Academic Press Limited
1076
H.-M. Jouve et al.
enzyme. However, the crystals of catalase A could be soaked in 10 mM-NADPH without any apparent, changes in the crystal parameters and symmetry. Crystals of catalase A, soaked in NADPH and then carefully washed four times with dinucleotide-free sulfate, could be dissolved in ammonium 100 mM-Tris buffer (pH 7.5). The enzyme) thus obtained, had the same biochemical characteristics as the B form: behavior in anion exchange chromatography and ability to reverse the complex II formation (Jouve et al., 1989). This suggested that NADPH was bound to the enzyme molecule in the crystal. CuKcl radiation was used with a conventional X-ray generator or with a rotating anode equipped with double mirrors to collimate the beam and separate diffraction spots corresponding to large cell parameters. The crystals diffract to 2.4 A resolution (1 A = 0.1 nm) and are stable in the X-ray beam for about 100 hours. X-ray precession photographs showed systematic reflection conditions on OOL, showed an hexagonal I, = 3n. HKO and HKI symmetry. and HOL, HlL, H2L showed two perpendicular mirror planes giving for the space groups either P6222 or its enantiomorph P6,22. The hexagonal unit cell parameters are n = b = 111.7 A. c = 249.8 A. A crystal density of 1.25 was found when measured on a gradient of carbon tetrachloride in water-saturated toluene (Matthews, 1968, 1985). From the volume of the unit cell, the number of tetrameric molecules per unit cell was calculated to be three and the solvent fraction 63%. Other possibilities are outside the range of reasonable values.
In space group P622. the number of eyuivalent positions is 12. consequently, the asymmetric unit contains one monomer. as for the M. lysodeikticus enzyme (Yusifov ef al., 1989). The catalasr of P. mirabiZia PR exhibits a true molecular 222 symmetry, whereas the symmetry is only nearIS 2-fold for the tetrameric catalase of beef liver and P. vitale (Melik-Adamyan et al., 1986). An initial data collection was ca,rried out at the L.C.R.E. synchrotron radiation facility on the 21) MARK2 electronic detector (Kahn et al.. 1986). Two crystals were used. one with c* along t,hr rotat*ion axis from 0” t)o 60” in phi and the &her with o* along the rotation axis beginning with (A* perpendicular to the X-ray beam (O” to 12” in phi). A tot,al of 48,842 diffract’ion intensities were collected to 2.9 a during the first c*ollection. giving 17.401 unique reflections after using the program PRO(‘OR of MADNES (Kabsch. 1988) in the space group P6222. This represent,s nearly 90(!,, of the complete data with an agreement factor I&,,,, = 4%(),, on fquivalent reflections and intensities. Since the unit, cell contains t#hree t,etrameric~ molrcules wit,h molecular spmmetry 222. t,he center of the tetramer must be on one of the positions of the P6*22 or (P6,22) cell with a 222 site1 symmetry. Moreover, the molecular axes P, &. R (Murbhy CJ~ trl.. 1981) must he aligned with the t,hree crystallo.graphic dyad axes intersecting atI this 222 sit)e. In each of the enantiomorphic spaccb groups, two different positions arc possible. namelv (0. 0. 0) or (l/2, 0. 0) with six different possible or&iations at each posit’ion. The R-factor. between observed and ~&uIated
Figure 1. Projection down the e2 axes. of the crystal structure of P. mirabiZi,s PR catalase The molecaular cent,rrs are positioned on the crystallographic sites with 222 symmetry at co-ordinates (l/‘L. 0. 0). (l/Z. l/2. 10). (0. 112, g/3). FIW solvent forms cylinders around the 6, axes with a diameter of 45 .+%shown in t,he Figure and channels orthogonal to the 6* axes (not shown).
Communications structure factors for data in the range 8 to 4 A, was computed with the program CORELS (Sussman, 1985) for each possible solution (co-ordinates of the molecular center and orientation), using as a model, the beef liver catalase (Fita & Rossmann, 1985) from the Protein Data Bank. In space group P6,22, R-factors for all possible solutions were approximately 56O/,. In space group P6222, centering the tetramer at (l/2, 0.0) with its P axis parallel to the 6-fold crystallographic axis and its & axis along a, gave an R-factor of 44%, while the other orientations and centerings gave an R-factor near 56O&. Using the beef liver catalase co-ordinates as a model for the P. mirabilis PR catalase, the packing of the molecules in the crystal is shown in Figure 1. Refinement and determination of the primary struct#ure are under
way.
References Beaumont, F.. Jouve, H. M., Gagnon, ,J., Gaillard, J. & Pelmont. J. (1990). Purification and properties of a catalase from potato tubers (Solanum tuberosum). Hunt A%. 72, 19-26. Deisseroth. A. & Dounce. A. L. (1970). Catalase: physical and chemical properties, mechanism of catalysis, and physiological role. Physiol. Rev. 50, 319-375. Fita. T. & Rossman. M. G. (1985). The NADPH binding site on beef liver catalase. Proc. Na.t. Acad. SC%., 1:S.A. 82, 1604-1608. Jouve, H. M., Lasaunikre, C. & Pelmont? J. (1983). Properties of a catalase from a peroxide-resistant mutant of Proteus mirabilis. Canad. J. Riochem. Cull Bid. 61. 1219.-1227. ,Jouve. H. M., Beaumont, F., Lkger, I.. Foray, J. & Pelmont. .J. (1989). Tightly bound KADPH in Proteus mirabilis catalase. Biochem. Cell Biol. 67: 27 I-277. Kattsh. EV. (1988). Evaluation of single crystal X-ray difli-action data from a position sensitive detector. J. Appl. C’rystallogr. 21. 916-934.
1077
Kahn, R., Fourme, R. & Bosshard, R. (1986). An areadetector diffractometer for the collection of high resolution and multiwavelength anomalous diffraction data in macromolecular crystallography. Nucl. Znstr. Meth. A246, 596-603. Kirkman, H. xi. & Gaetani, G. F. (1984). Catalase: a tetrameric enzyme with four tightly bound molecules Proc. Nut. ilcad. Sci.. I:.S.A. 81, of KADPH. 4343-4347. Kirkman, H. N., Galiano, S. & Gaetani, G. F. (1987). The function of catalase-bound NADPH. ./. Kiol. Chem. 262. 660-666.
Matthews, B. W. (1968). Solvent content of protein crystals. d. Mol. Biol. 33. 491-497. Matthews, B. W. (1985). Determination of protein molecular weight, hydration, and packing from crystal density. In Methods in &zymology (Colowick. S. P. & Kaplan, N. 0.. eds), vol. 114. pp. 176-187. Academic Press. New York. Melik-Adamyan, W. R.. Barynin, V. V.. Vagin, A. A.. Borisov. V. V.. Vainshtein. B. K.. Fita. I.. Murthp. M. R. N. & Rossmann, M. G. (I 986). Comparison of beef liver and Penkillium vita/e catalasrs. J. Mol. Riol. 188. 63-72. Murthy. II. R. N;., Reid. T. ,J.. III, Sicignano. A., Tanaka. N. & Rossman, M. G. (1981). St’ruc*turc> of beef liver catalase. ,I. Mol. Biol. 152. 465-499. Sussman, ,J. L. (1985). Constrained-restrained leastsquares ((IORELS) refinement of proteins and nucleic (Colowick, S. P. & acids. In Methods in &zymology Kaplan. S. 0.: eds), vol. 11.5, pp. 271.-303. Academic, Press. &w York. Vainshtrin. B. K.. Melik-Adamyan. W. R.. Barynin. V. V., Vagin, A. A., Grebenko. .4. T.. Borisov. 3. V.. Bartels. K. S.. Fita. I. & Rossman. M. G. (1986). Three-dimensional of ratalase structure from Penicilliu,m vitale at 2.0 p\ resolut,ion. .I. ~Vol. Biol. 188. 49-61. Yusifov. E. F.. Grebenko, 9. I.. Barynin, V. V.. Murshudov. G. E;., Vagin. A. A.. Melik-Adamyan. V. R. & Vainshtein. B. K. (1989). Three-dimensional structure of ratalase from Micrococczts lysodeikticus at a resolution of 3.0 A. SOT:. Phys. Cry.stallogr. 34. 870-854.
Edited by R. Huber
