Deoxyribose-5-phosphate Aldolase

Crystallization

50. Stura E.A, Ghosh S, Garcia-Junceda E, Chen L, Wong C-H, Wilson I.A. (1995) Crystallization and preliminary crystallographic data for class I Deoxy-5-Phosphate aldolase from Escherichia coli: An Application of Reverse Screening. Proteins, 22:67-72.

Abstract: X-ray quality crystals of class I deoxyribose-5-phosphate aldolase from Escherichia coli have been obtained for the unliganded enzyme and in complex with its substrate, 2-deoxyribose-5-phosphate. The enzyme catalyzes the reversible cleavage of 2-deoxyribose-5-phosphate to acetaldehyde and D-glyceraldehyde-3-phosphate.The unliganded and complex crystals are prismatic long rods and belong to the orthorhombic space group P212121 with cell dimensions a = 183.1 Å, b = 61.4 Å, c = 49.3 Å and a = 179.2 Å, b = 60.5, Å, c = 49.1 Å, respectively. Two molecules in the asymmetric unit are related by a non-crystallographic 2-fold axis. The crystals are stable in the X-ray beam and diffract to at least 2.6 Å. A new method, reverse screening, designed to minimize protein utilization during the screening process was used to determine supersaturation and crystallization conditions.

Crystallization conditions For Aldolases:

A Survey of the BMCD database

Crystallization conditions Cell parameters Reference
3.3M ammonium sulfate, pH 6.0
2.3M ammonium sulfate, 20mM KH2PO4, 1mM NH4OH, .1 mM EDTA, pH 6		 P6222 a = b = 96.2 Å c = 168.0 Å 1
5
16% PEG 6,000, 100mM Tris-HCl, 1mM EDTA, 1mM bME, 0.5% garamycin, pH 7.8, 2-4°C P212121 a = 86.3 Å b = 115.7 Å , c = 151.4 Å 2
50-55% ammonium sulfate, pH 6.1-6.4, 2-3°C
2.3M ammonium sulfate, 100mM citrate, 5 mM EDTA, pH 6.0, 4°C		 P6222 a = b = 161.0 Å, c = 170.0 Å 3
4,5
40-49% ammonium sulfate, 100mM TEA, 5% EDTA, pH 7.3 , 20°C (and with citrate buffer pH 5.5 P21 a = 164.5 Å, b = 57.3 Å, c = 85.0 Å beta=102.7° 4
2M NaH2PO4, K2HPO4, pH 6.0-7.5.
50-55% ammonium sulfate, pH 6.1-6.4, 2-3°C.		 P6222 a = b = 163.5 Å, c = 335.0 Å 6,3
45% ammonium sulfate, 0.1 M TEA-HCl , pH 7.4, 4°C C2221 a = 377.0 Å, b = 130.3 Å, c = 80.0 Å 7
20-40mM KH2PO4, 0-0.2 M ammonium sulfate, pH 3.5, 24°C P213 a = b = c = 103.5 Å 8
7% PEG 4,000, 30 mM Tris-HCl,2.5 mM Zn Cl2, pH 7.5 P6122 a = b = 78.4 Å, c = 290.6 Å 9
20% MPEG 5,000, 200 mM imidazole malate, pH 6.0 P212121 a = 183.1 Å b = 61.4 Å , c = 43.9 Å This Work
25% MPEG 2,000, 200 mM imidazole malate,250 mM LiCl, pH 5.8 and 3.8 mM 2-deoxyribose 5-phosphate P212121 a = 179.2 Å b = 60.5 Å , c = 49.1 Å This Work

Crystallization conditions in the BMCD database for :

fructose-1,6-bisphosphate aldolases from human :

[1] Millar, J. R., Shaw, P. J., Stammers, D. K., Watson, H. C. The low resolution structure of human muscle aldolase. Phil. Trans. Roy. Soc. Lond. B 293:209-214, 1981.

Drosophila melanogaster:

[2] Brenner-Holzach, 0., Smit, J. D. G. Crystallization and preliminary crystallographic data for fructose-1,6-bisphosphate aldolase from Drosophila melanogaster. J. Biol. Chem. 257:11747-11749, 1982.

bovine:

[3] Goryunov, A. K., Andreeva, N. S., Spitsberg, V. L. Biofizika 14:1116-1117, 1969.

rabbit:

[4] Eagles, P. A. M., Johnson, L. N., Joynson, M. A., McMurray, C. H., Gutfreund, H. Subunit structure of aldolase: chemical and crystallographic evidence. J. Mol. Biol. 45:533-544, 1969.
[5] Heidner, E. G., Weber, B. H., Eisenberg, D. Subunit structure of aldolase. Science 171:677-680, 1971.
[6] Sawyer, L. A fourth crystal form of rabbit muscle aldolase. J. Mol. Biol. 71:503-505, 1972.
[7] Sygusch, J. & Beaudry, D. Preliminary crystallographic investigation of rabbit liver aldolase. J. Mol. Biol. 186: 215-217, 1985.

phospho-2-keto-3-deoxy-gluconate aldolase from Pseudomonas putida

[8] Vandlen, R. L., Ersfeld, D. L., Tulinsky, A. , Wood, W. A. Confirmation of a trimeric subunit arrangement for 2-keto-3-deoxy-6-phosphogluconic aldolase using x-ray crystallographic methods. J. Biol. Chem. 248:2251-2253, 1973.

The crystallization conditions for the E. coli fructose-1,6-bisphosphate aldolase ([9] at 18 mg/ml):

[9] Naismith, J. H., Ferrara, J. D., Bailey, S., Marshall, K., Dauter, Z., Wilson, K. S., Habash, J., Harrop, S. J., Berry, A. J., Hunter, W. N. Initiating a crystallographic study of a class II fructose-1,6-bisphosphate aldolase. J. Mol. Biol. 225:1137-1141, 1992.
were not utilized in the reverse screening since these were not in the BMCD (version 2.1). For comparison, the crystallization conditions obtained for the E. coli FDPA (at 10 mg/ml instead of 18 mg/ml), with the same precipitant, are:

25-28% PEG 4,000 (or MPEG 2,000), 0.2 M imidazole malate, pH 5.5-6.2