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Preliminary X-ray crystallographic study of lysozyme produced by Streptomyces globisporus
J. Mol. Biol. (1989) 207, 851-852
Preliminary X-ray Crystallographic Study of Lysozyme Produced by Streptomyces globisporus Lysozyme from Streptomyces globisporus has been crystallized in a form suitable for X-ray structure analysis using ammonium sulfate as a precipitant. The crystals are hexagonal, space group P6,22 (P6,22) with unit cell dimensions: a = b = 129 A, c = 143 A. There are three or four molecules per asymmetric unit. The crystals diffract X-rays to at least 3.0 a resolution.
Lysozyme, which was originally discovered by Fleming, is the enzyme which causes lysis of cell walls of bacteria, such as Mycrococcus lysodeikticus, by hydrolyzing the /?-(1 + 4)-glycosidic bonds of the polysaccharide backbone of the peptidoglycan, and it is well known that the enzyme is widespread in animals, plants and micro-organisms. On the basis of the homology of amino acid sequence, !ysozymes whose amino acid sequences are known are classified into four distinct types: (1) chicken, (2) phage, (3) goose, and (4) Chalaropsis. Among lysozymes, hen egg-white lysozyme (HEWL,? 129 residues) is the most extensively investigated and its amino acid sequence was determined independently by Joll& et aE. (1963) and Canfield (1963). Lysozymes from vertebrates such as birds, human, baboon and rat are related to HEWL (for a review, see Imoto et al., 1972; Hamaguchi & Hayashi, 1978). On the other hand, embden goose (GEWL; Simpson & Morgan, 1983) and black swan (SEWL; Simpson et al., 1980) produce a form of lysozyme in their egg-white different from the chicken type. They contain more amino acid residues (185 residues) than HEWL and do not cross-react immunologically with the chicken-type lysozymes (Arnheim et al., 1973). The phage type includes lysozymes from bacteriophages T4 (T4L, 164 residues; Tsugita & Inouye, 1968) and T2 (T2L, 164 residues; Inouye & Tsugita, 1968). Lysozymes of the Chalaropsis type are extracted from ChaEaropsis species (CHL, 211 residues; Felch et al., 1975) and Streptomyces erythraeus (SEL, 202 residues; Hara et aE., unpublished results. Although there is no obvious sequence homology between one class and another, X-ray structure analyses of HEWL (Blake et al., 1965), T4L (Remington et al., 1978) and GEWL (Griitter et al., 1983) indicate that three-dimensional structures of these three types of lysozyme are similar to one another. Moreover, a detailed comparison of the three-dimensional struct,ures suggested that these three classes of lysozyme
common evolutionary precursor (Rossman & Argos, 1976; Matthews et al., 1981; Griitter et al., 1983; Weaver et al., 1985). On the other hand, the amino acid sequence and also the three-dimensional structure of SEL show no obvious similarities to other lysozymes (Harada et aE., 1981), indicating that lysozymes of the Chalaropspsis type are not evolutionarily related to the other lysozymes. Streptomyces globisporus produces two kinds of lysozyme (M-l and M-2 lysozyme) and secretes them in the cultural broth. The molecular weights are about 20,000 and 11,000 for M-l and M-2 lysozyme, respectively. Analysis of the amino acid composition of M-l and M-2 lysozyme indicated that (1) M-l type, and lysozyme belongs to the Chdaropsis (2) M-2 lysozyme is similar to HEWL but there was no cysteine restidue in this enzyme (Kawata et al., 1983). Here we report preliminary crystallographic data of the crystals of M-l lysozyme. Electrophoretically pure M-l lysozyme was isolated from mutanolysin, which was kindly supplied by Dainippon Pharmaceutical Corp. Ltd (Osaka, Japan), according to published procedures (Yokogawa et al., 1975). The crystals were grown from ammonium sulfate by two methods, the hanging drop vapor diffusion method and the microdialysis method. The first method, which gave many small crystals, was used to check the range of possible crystallization conditions. The large crystals suitable for X-ray analysis were obtained by the second method. About 10 mg of the lyophilized protein were dissolved in 300 ~1 of distilled water and undissolved material was removed by centrifugation. The resulting clear protein solution was dialyzed in microdialysis cells against 50 mM-imidazol*HCl buffer (pH 7.5) containing 40% saturated ammonium sulfate. Long hexagonal rod-shaped crystals with a typical size of 1.0 mm x0.3 mm x0-3 mm grew within a week. When the concentration of ammonium sulfate was
(chicken.
lower
phage
and goose types)
diverged
from
a
or higher
than
40%,
long
fine
crystals
or
many small crystals grew, respectively. The X-ray diffraction patterns were recorded with an EnrafNonius precession camera (Ni-filtered CuKcr radiation from a rotating anode X-ray generator). The crystals belong to the hexagonal space group P6,22
t Abbreviations used: HEWL, hen egg-white lysozyme; SEWL, swan egg-white lysozyme, GEWL, goose egg-white lysozyme; SEL, Streptomyces erythraeus lysozyme: CHL. Ch&zropsis lysozyme.
851 0022-2836B9/12085lb4B
$03.00/O
0
1989 Academic
Press Limited
852
S. Harada
(P6,22), with unit cell dimensions: a = b = 1298, c= 143w and V=2*06x106A3 (l~%=O.lnm). This gives values for V, of 2.86 A3/dalton asymmetric unit) or (20,000 M, X 3 per 2.15 A3/dalton (20,000 M, x 4 per asymmetric unit); both values are in a range that is normally found in protein crystals (Matthews, 1968). The crystals are stable to X-ray irradiation and diffract to at least 3.0 L%resolution. Three heavy-atom derivatives have been obtained by using methylmercury chloride, mersalyl and dipotassium tetrachloroplatinate. A search for other heavy-atom derivatives and data collection from native crystals is in progress.
Shigeharu Harada Kengo Kitadokoro Takahide Fujii Yasushi Kai Nobutami Kasai Department of Applied Chemistry Faculty of Engineering Osaka University Suita, Osaka 565, Japan
et al. Felch, ,J. W., Inagami, T. & Hash, J. H. (1!175). .I. Hiol. Chem. 250, 3713-3720. Griitter, M. G., Weaver, L. H. & Matthews, B. W. (1983). Nature (London), 303, 828-83 1. Hamaguchi, K. & Hayashi, K. (1978). Molecular Ba&s oj Enzyme Function - Lysozyme, Kodansha. Tokyo. Harada, S., Sarma, R., Kakudo, M.. Hara. S. bz Ikenaka. T. (1981). .J. Biol. Chem. 256, 116OC-11602. Imoto, T., Johnson, I,. N.. Xorth, A. (1. T., Phillips, I). (‘. & Rupley, tJ. A. (1972). In The h’nzymecs (Boyer. P. D., ed.). vol. 7. 3rd edit.. pp. 66.5868, Academiv Press, New York. Inouye, M. & Tsugita, A. (1968). J. Mol. Hiol. 37. 21% 223. ~Joll8s. ,J.; ,Jauregui-Adell, J.. Bernier, I. 8 ,Joll&. I’. (1963). Biochim. Biophys. Acta, 78, 66fG-689. Kawata. S.. Takemura, T. & Yokogawa, K. (I 983). Ayric. Biol. Chem. 47, 1501-1508. Matthews, B. W. (1968). J. Mol. Biol. 33. G--497. Matthews, B. W., Griitter. M. G.. Anderson, W. F. & Remington, S. ,J. (1981). Nature (London), 290, 334. 335. Remington, S. J., Anderson, W. P.. Owen, .I., Ten Eyke. L,. F., Grainger, C. T. & Matthews. B. W. (1978). J. Mol. Biol. 118, 81-98. Rossmann. M. G. & Argos, P. (1976). .I. %ol. Hiol. 105.
75-95.
Received 19 December 1988, and in revised form 2 March 1989
References Arnheim, N., Hidenburg, A., Begg, G. S. & Morgan, F. J. (1973). J. Biol. Chem. 248, 8036-8042. Blake, C. C. F., Koenig, D. F., Mair, G. A., North, A. C. T., Phillips, D. C. & Sarma, V. R. (1965). Nature (London), 206, 757-763. Canfield, R. E. (1963). J. Riol. Chem. 238, 2698-2707.
Edited
Simpson, R. J. & Morgan, F. J. (1983). Hiochim. &qphyys. Acta, 744, 349-351. Simpson, R. J., Begg, G. S., Dorow. D. S. & Morgan. F. J. (1980). Biochemistry, 19, 1814--1819. Tsugita, A. & Inouye. M. (1968). .I. Mol. Riol. 37. 201.212. Weaver, L. H.. Griitter, M. G.. Remington, S. .J., Gray, T. M., Isaacs. N. W. & Matthews, R. W. (1985). J. Mol. Evol. 21. 97-111. Yokogawa, K., Kawata, S., Takemura. ‘I’. & Yoshimura. Y. (1975). Agric. Biol. Chcm. 39, 1533-1543.
by R. Huber
Preliminary X-ray Crystallographic Study of Lysozyme Produced by Streptomyces globisporus Lysozyme from Streptomyces globisporus has been crystallized in a form suitable for X-ray structure analysis using ammonium sulfate as a precipitant. The crystals are hexagonal, space group P6,22 (P6,22) with unit cell dimensions: a = b = 129 A, c = 143 A. There are three or four molecules per asymmetric unit. The crystals diffract X-rays to at least 3.0 a resolution.
Lysozyme, which was originally discovered by Fleming, is the enzyme which causes lysis of cell walls of bacteria, such as Mycrococcus lysodeikticus, by hydrolyzing the /?-(1 + 4)-glycosidic bonds of the polysaccharide backbone of the peptidoglycan, and it is well known that the enzyme is widespread in animals, plants and micro-organisms. On the basis of the homology of amino acid sequence, !ysozymes whose amino acid sequences are known are classified into four distinct types: (1) chicken, (2) phage, (3) goose, and (4) Chalaropsis. Among lysozymes, hen egg-white lysozyme (HEWL,? 129 residues) is the most extensively investigated and its amino acid sequence was determined independently by Joll& et aE. (1963) and Canfield (1963). Lysozymes from vertebrates such as birds, human, baboon and rat are related to HEWL (for a review, see Imoto et al., 1972; Hamaguchi & Hayashi, 1978). On the other hand, embden goose (GEWL; Simpson & Morgan, 1983) and black swan (SEWL; Simpson et al., 1980) produce a form of lysozyme in their egg-white different from the chicken type. They contain more amino acid residues (185 residues) than HEWL and do not cross-react immunologically with the chicken-type lysozymes (Arnheim et al., 1973). The phage type includes lysozymes from bacteriophages T4 (T4L, 164 residues; Tsugita & Inouye, 1968) and T2 (T2L, 164 residues; Inouye & Tsugita, 1968). Lysozymes of the Chalaropsis type are extracted from ChaEaropsis species (CHL, 211 residues; Felch et al., 1975) and Streptomyces erythraeus (SEL, 202 residues; Hara et aE., unpublished results. Although there is no obvious sequence homology between one class and another, X-ray structure analyses of HEWL (Blake et al., 1965), T4L (Remington et al., 1978) and GEWL (Griitter et al., 1983) indicate that three-dimensional structures of these three types of lysozyme are similar to one another. Moreover, a detailed comparison of the three-dimensional struct,ures suggested that these three classes of lysozyme
common evolutionary precursor (Rossman & Argos, 1976; Matthews et al., 1981; Griitter et al., 1983; Weaver et al., 1985). On the other hand, the amino acid sequence and also the three-dimensional structure of SEL show no obvious similarities to other lysozymes (Harada et aE., 1981), indicating that lysozymes of the Chalaropspsis type are not evolutionarily related to the other lysozymes. Streptomyces globisporus produces two kinds of lysozyme (M-l and M-2 lysozyme) and secretes them in the cultural broth. The molecular weights are about 20,000 and 11,000 for M-l and M-2 lysozyme, respectively. Analysis of the amino acid composition of M-l and M-2 lysozyme indicated that (1) M-l type, and lysozyme belongs to the Chdaropsis (2) M-2 lysozyme is similar to HEWL but there was no cysteine restidue in this enzyme (Kawata et al., 1983). Here we report preliminary crystallographic data of the crystals of M-l lysozyme. Electrophoretically pure M-l lysozyme was isolated from mutanolysin, which was kindly supplied by Dainippon Pharmaceutical Corp. Ltd (Osaka, Japan), according to published procedures (Yokogawa et al., 1975). The crystals were grown from ammonium sulfate by two methods, the hanging drop vapor diffusion method and the microdialysis method. The first method, which gave many small crystals, was used to check the range of possible crystallization conditions. The large crystals suitable for X-ray analysis were obtained by the second method. About 10 mg of the lyophilized protein were dissolved in 300 ~1 of distilled water and undissolved material was removed by centrifugation. The resulting clear protein solution was dialyzed in microdialysis cells against 50 mM-imidazol*HCl buffer (pH 7.5) containing 40% saturated ammonium sulfate. Long hexagonal rod-shaped crystals with a typical size of 1.0 mm x0.3 mm x0-3 mm grew within a week. When the concentration of ammonium sulfate was
(chicken.
lower
phage
and goose types)
diverged
from
a
or higher
than
40%,
long
fine
crystals
or
many small crystals grew, respectively. The X-ray diffraction patterns were recorded with an EnrafNonius precession camera (Ni-filtered CuKcr radiation from a rotating anode X-ray generator). The crystals belong to the hexagonal space group P6,22
t Abbreviations used: HEWL, hen egg-white lysozyme; SEWL, swan egg-white lysozyme, GEWL, goose egg-white lysozyme; SEL, Streptomyces erythraeus lysozyme: CHL. Ch&zropsis lysozyme.
851 0022-2836B9/12085lb4B
$03.00/O
0
1989 Academic
Press Limited
852
S. Harada
(P6,22), with unit cell dimensions: a = b = 1298, c= 143w and V=2*06x106A3 (l~%=O.lnm). This gives values for V, of 2.86 A3/dalton asymmetric unit) or (20,000 M, X 3 per 2.15 A3/dalton (20,000 M, x 4 per asymmetric unit); both values are in a range that is normally found in protein crystals (Matthews, 1968). The crystals are stable to X-ray irradiation and diffract to at least 3.0 L%resolution. Three heavy-atom derivatives have been obtained by using methylmercury chloride, mersalyl and dipotassium tetrachloroplatinate. A search for other heavy-atom derivatives and data collection from native crystals is in progress.
Shigeharu Harada Kengo Kitadokoro Takahide Fujii Yasushi Kai Nobutami Kasai Department of Applied Chemistry Faculty of Engineering Osaka University Suita, Osaka 565, Japan
et al. Felch, ,J. W., Inagami, T. & Hash, J. H. (1!175). .I. Hiol. Chem. 250, 3713-3720. Griitter, M. G., Weaver, L. H. & Matthews, B. W. (1983). Nature (London), 303, 828-83 1. Hamaguchi, K. & Hayashi, K. (1978). Molecular Ba&s oj Enzyme Function - Lysozyme, Kodansha. Tokyo. Harada, S., Sarma, R., Kakudo, M.. Hara. S. bz Ikenaka. T. (1981). .J. Biol. Chem. 256, 116OC-11602. Imoto, T., Johnson, I,. N.. Xorth, A. (1. T., Phillips, I). (‘. & Rupley, tJ. A. (1972). In The h’nzymecs (Boyer. P. D., ed.). vol. 7. 3rd edit.. pp. 66.5868, Academiv Press, New York. Inouye, M. & Tsugita, A. (1968). J. Mol. Hiol. 37. 21% 223. ~Joll8s. ,J.; ,Jauregui-Adell, J.. Bernier, I. 8 ,Joll&. I’. (1963). Biochim. Biophys. Acta, 78, 66fG-689. Kawata. S.. Takemura, T. & Yokogawa, K. (I 983). Ayric. Biol. Chem. 47, 1501-1508. Matthews, B. W. (1968). J. Mol. Biol. 33. G--497. Matthews, B. W., Griitter. M. G.. Anderson, W. F. & Remington, S. ,J. (1981). Nature (London), 290, 334. 335. Remington, S. J., Anderson, W. P.. Owen, .I., Ten Eyke. L,. F., Grainger, C. T. & Matthews. B. W. (1978). J. Mol. Biol. 118, 81-98. Rossmann. M. G. & Argos, P. (1976). .I. %ol. Hiol. 105.
75-95.
Received 19 December 1988, and in revised form 2 March 1989
References Arnheim, N., Hidenburg, A., Begg, G. S. & Morgan, F. J. (1973). J. Biol. Chem. 248, 8036-8042. Blake, C. C. F., Koenig, D. F., Mair, G. A., North, A. C. T., Phillips, D. C. & Sarma, V. R. (1965). Nature (London), 206, 757-763. Canfield, R. E. (1963). J. Riol. Chem. 238, 2698-2707.
Edited
Simpson, R. J. & Morgan, F. J. (1983). Hiochim. &qphyys. Acta, 744, 349-351. Simpson, R. J., Begg, G. S., Dorow. D. S. & Morgan. F. J. (1980). Biochemistry, 19, 1814--1819. Tsugita, A. & Inouye. M. (1968). .I. Mol. Riol. 37. 201.212. Weaver, L. H.. Griitter, M. G.. Remington, S. .J., Gray, T. M., Isaacs. N. W. & Matthews, R. W. (1985). J. Mol. Evol. 21. 97-111. Yokogawa, K., Kawata, S., Takemura. ‘I’. & Yoshimura. Y. (1975). Agric. Biol. Chcm. 39, 1533-1543.
by R. Huber