,I. Mol. Riol. (1976) 102, 173-175
Preliminary Crystallographic
Data for Beta-lytic Protease
Btlta-lytic protease from Myzobacter 495 was crystallized by dialysis wit,h 1 &I-sodium chloride and 0.1 M-sodium citrate (pH 5.95). The crystals werr rhombic prisms with space group P212,2, and unit cell parameters a = 54.1, 71 :- 99.6 a.nd c = 53.9 A. Considerations of tbc call volun~e and moleclllar \vczi&t indicate two molecules of beta-lyt,ic proteasc in tile as>-mmrtric Illlit. Beta-lytic protease is an extracellular neutral metallo-protease which was isolated (Whitaker, 1965) from culture filtrates of the soil bacillus Nyxobacter 495. It has a molecular weight of 19,200 wit)h one atom of zinc per molecule (Jurasek & Whitaker, 1966). The enzyme is an endopeptidase with a marked preference for cleaving peptide linkages which have a glycyl residue as donor of the -CO. group and/or a residue of a hydrophobic amino acid as donor of the .NHgroup (Allen. 1973). Its bacteriolytic activity stems from its ability to cleave certain cross-linkages a’nd linkages from muramic acid in the peptidoglycan network of bacterial cell walls (Tsai et al.. 1965). The amino acid sequence of beta-lytic protease (Damaglou et al., 1976) shows no The two enzymes are probably significant’ homology with that of thermolysin. products of different pathways in the evolution of bacterial neut’ral proteases. A similar relationship exists between the subtilisin group of bacterial serine proteases and another bacteriolytic protease of Myxobacter 495, alpha-lytic protease. which is clearly a homologue of the pancreatic serine proteases (Olson et al., 1970). The crystallization propert’ies of beta-lytic proteasc were investigated using t.he equilibrium dialysis technique of Zeppezauer ef al. (1968). Purified enzyme was prepared hp the procedure of Whitaker (1967). Enzyme solutions were prepared hy dissolving 10 mg of freeze-dried enzyme in 1 ml of deionized distilled wat,er. This solution was sealed in glass capillaries (1.2 mm i.d.) with dialysis tubing and dialyzed at room temperature with 1 M-NaCl and O-1 M-sodium citrate adjusted to pH 5.95 with HCl. Thin diamond-shaped platelets appeared u-it’hin 24 hours and rhombic prisms within an additional 72 hours. Both cryst)al forms were st’able only wit,hin the pH range 5.8 to 6.2 in t’he presence of citrate. Typical dimensions for t’he two crystal forms were 0.2 mm r’ (1.2 mm x 0.01 mm for the plat,elets and 0.2 mm xl 0.3 mm Y 0.5 mm for the prisms. Thta rhombic prisms exhibited an intense diffraction pattern extending beyond a resolution of 2.5 A. The space group was I>212,2, with unit, cell parameters a = 54.1. h = 99.6. c = 53.9 A and I’ = 290.400 A3. Th e crystal density, measured in a chlorot)enzene/hromobenzene gradient. was 1.26 p/cm3. From the crystallizatSiou liquor density of 1.106 g/cm3. and an assumed value of 0.74 cm3/g for the partial specific volume of the protein, the unit cell contains eight enzyme molecules with about 35:; of the cell volume occupied by solvent,. The T-, ratio of 1.89 A3/dalton is within the range of values (Matthews, 1968) for ot’her crystalline globular proteins. The platelets. which exhibited a weak diffraction pattern observa,b]e only to a resolution of 4 w. were orthorhombic, space group (‘222 or (T222, with unit cell 153
174
W. B. T. CRUSE
FIG. 1. Rhombic prism modification parallel to the c-axis.
ASI)
I).
Ii.
of hot:%-lytic protoasc.
FIQ. 2. A p = 16” precession photograph filtered Cu Ka radiation at 15 mA.
\VHI’L’AKEH
l’ho long dimension
of the Ok1 zone. The exposure
(- 0.5 mm) is
is for 24 h with
Ni-
parameters a = 104.7, b = 89.8 and c = 95.9 A. The Vm ratio for 16 molecules in the unit cell was 2.94 d3/dalton. Three heavy-atom isomorphous derivatives of the rhombic prism modification NaAuCl, and K,PtCI,, respectively. Threehave been prepared with K&Cl,, dimensional intensity data for derivative and native crystals are currently being collected to a resolution of 3.5 A.
LETTERS
TO THE
17:x
EDITOR
This work was supported by the Medical Research Council of Canada. We thank National Research Council of Canada for the use of X-ray diffraction equipment,.
W. B. T. CRUSE D. R. WHITAKER
Department of Biochemistry Ottawa Universit> Ottawa, Ontario, Canada Rcceivlxl
6 October
the
1975 REFERENCES
Allen, L. C. (1973). Ph.D. Thesis, Ottawa University, Ottawa, Ontario, Canada. Damaglou, A. P.. Allen, L. C. & Whitaker, D. R. (1976). In Atlas of Protein Sequence and Structure (Dayhoff, M. O., ed.), vol. 5, suppl. 2, National Biomedical Research Foundation, Washington, in the press. .Jurasek, L. & Whitaker, D. R. (1966). Can. J. &o&em. 45, 917-927. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Olson, M. 0. J., Nagabushan, N., Dzwiniel, M., Smillio, L. B. & Whitaker, D. R. (1970). Nature (London), 228, 438-442. Tsai, C. S., Whitaker, D. R., Jurasek, L. & Gillespie, J>. (11.(1965). Can. J. Biochem. 43, 1971l1983. Whitaker, D. R. (1965). Can. J. Biochem. 43, 19351954. Whitaker, D. R. (1967). Can. J. Biochem. 45, 991-993. Zeppezauer, M., Eklund, H. & Zeppezauer, E. S. (1968). Arch. &o&em. Biophys. 123, 564-573.
Preliminary Crystallographic
Data for Beta-lytic Protease
Btlta-lytic protease from Myzobacter 495 was crystallized by dialysis wit,h 1 &I-sodium chloride and 0.1 M-sodium citrate (pH 5.95). The crystals werr rhombic prisms with space group P212,2, and unit cell parameters a = 54.1, 71 :- 99.6 a.nd c = 53.9 A. Considerations of tbc call volun~e and moleclllar \vczi&t indicate two molecules of beta-lyt,ic proteasc in tile as>-mmrtric Illlit. Beta-lytic protease is an extracellular neutral metallo-protease which was isolated (Whitaker, 1965) from culture filtrates of the soil bacillus Nyxobacter 495. It has a molecular weight of 19,200 wit)h one atom of zinc per molecule (Jurasek & Whitaker, 1966). The enzyme is an endopeptidase with a marked preference for cleaving peptide linkages which have a glycyl residue as donor of the -CO. group and/or a residue of a hydrophobic amino acid as donor of the .NHgroup (Allen. 1973). Its bacteriolytic activity stems from its ability to cleave certain cross-linkages a’nd linkages from muramic acid in the peptidoglycan network of bacterial cell walls (Tsai et al.. 1965). The amino acid sequence of beta-lytic protease (Damaglou et al., 1976) shows no The two enzymes are probably significant’ homology with that of thermolysin. products of different pathways in the evolution of bacterial neut’ral proteases. A similar relationship exists between the subtilisin group of bacterial serine proteases and another bacteriolytic protease of Myxobacter 495, alpha-lytic protease. which is clearly a homologue of the pancreatic serine proteases (Olson et al., 1970). The crystallization propert’ies of beta-lytic proteasc were investigated using t.he equilibrium dialysis technique of Zeppezauer ef al. (1968). Purified enzyme was prepared hp the procedure of Whitaker (1967). Enzyme solutions were prepared hy dissolving 10 mg of freeze-dried enzyme in 1 ml of deionized distilled wat,er. This solution was sealed in glass capillaries (1.2 mm i.d.) with dialysis tubing and dialyzed at room temperature with 1 M-NaCl and O-1 M-sodium citrate adjusted to pH 5.95 with HCl. Thin diamond-shaped platelets appeared u-it’hin 24 hours and rhombic prisms within an additional 72 hours. Both cryst)al forms were st’able only wit,hin the pH range 5.8 to 6.2 in t’he presence of citrate. Typical dimensions for t’he two crystal forms were 0.2 mm r’ (1.2 mm x 0.01 mm for the plat,elets and 0.2 mm xl 0.3 mm Y 0.5 mm for the prisms. Thta rhombic prisms exhibited an intense diffraction pattern extending beyond a resolution of 2.5 A. The space group was I>212,2, with unit, cell parameters a = 54.1. h = 99.6. c = 53.9 A and I’ = 290.400 A3. Th e crystal density, measured in a chlorot)enzene/hromobenzene gradient. was 1.26 p/cm3. From the crystallizatSiou liquor density of 1.106 g/cm3. and an assumed value of 0.74 cm3/g for the partial specific volume of the protein, the unit cell contains eight enzyme molecules with about 35:; of the cell volume occupied by solvent,. The T-, ratio of 1.89 A3/dalton is within the range of values (Matthews, 1968) for ot’her crystalline globular proteins. The platelets. which exhibited a weak diffraction pattern observa,b]e only to a resolution of 4 w. were orthorhombic, space group (‘222 or (T222, with unit cell 153
174
W. B. T. CRUSE
FIG. 1. Rhombic prism modification parallel to the c-axis.
ASI)
I).
Ii.
of hot:%-lytic protoasc.
FIQ. 2. A p = 16” precession photograph filtered Cu Ka radiation at 15 mA.
\VHI’L’AKEH
l’ho long dimension
of the Ok1 zone. The exposure
(- 0.5 mm) is
is for 24 h with
Ni-
parameters a = 104.7, b = 89.8 and c = 95.9 A. The Vm ratio for 16 molecules in the unit cell was 2.94 d3/dalton. Three heavy-atom isomorphous derivatives of the rhombic prism modification NaAuCl, and K,PtCI,, respectively. Threehave been prepared with K&Cl,, dimensional intensity data for derivative and native crystals are currently being collected to a resolution of 3.5 A.
LETTERS
TO THE
17:x
EDITOR
This work was supported by the Medical Research Council of Canada. We thank National Research Council of Canada for the use of X-ray diffraction equipment,.
W. B. T. CRUSE D. R. WHITAKER
Department of Biochemistry Ottawa Universit> Ottawa, Ontario, Canada Rcceivlxl
6 October
the
1975 REFERENCES
Allen, L. C. (1973). Ph.D. Thesis, Ottawa University, Ottawa, Ontario, Canada. Damaglou, A. P.. Allen, L. C. & Whitaker, D. R. (1976). In Atlas of Protein Sequence and Structure (Dayhoff, M. O., ed.), vol. 5, suppl. 2, National Biomedical Research Foundation, Washington, in the press. .Jurasek, L. & Whitaker, D. R. (1966). Can. J. &o&em. 45, 917-927. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Olson, M. 0. J., Nagabushan, N., Dzwiniel, M., Smillio, L. B. & Whitaker, D. R. (1970). Nature (London), 228, 438-442. Tsai, C. S., Whitaker, D. R., Jurasek, L. & Gillespie, J>. (11.(1965). Can. J. Biochem. 43, 1971l1983. Whitaker, D. R. (1965). Can. J. Biochem. 43, 19351954. Whitaker, D. R. (1967). Can. J. Biochem. 45, 991-993. Zeppezauer, M., Eklund, H. & Zeppezauer, E. S. (1968). Arch. &o&em. Biophys. 123, 564-573.
