T. Skálová1, J. Hašek1, H. Petroková1, E. Buchtelová1, J. Dohnálek1, P. Lipovová2, V. Spiwok2, H. Strnad2 and B. Králová2


1 Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, 162 06 Praha 6, Czech Republic

2 Institute of Chemical Technology, Technická 5, 166 28 Praha 6, Czech Republic


The β-galactosidase from Arthrobacter sp. C2-2, main subject of this study,  was derived from bacteria living in Antarctica under low temperatures (permanently below 5°C). Its low temperature activity deserves special attention because of many practical and theoretical impacts of enzymatic processes under ambient temperatures [1,2]. The elucidation of low temperature adaptation of enzymes requires knowledge of detailed molecular geometry and an analysis of conformational changes in the active site. The only known method being able to give complete determination of structure of large proteins is the X-ray crystallography. Therefore, the first part of our project  is to determine the structure the above mentioned cold-active enzyme using X-ray diffraction.

Cold active β-galactosidase from Arthrobacter sp. C2-2 is a large protein - homotetramer with molecular weight 500 kDa, having 4092 residues. It is homological to the β-galactosidase from Escherichia coli (sequence identity 32%). Expression, purification and basic characteristics of the protein performed in the institute of Chemical Technology in Praha were published in [1,2]. The protein was crystallized using hanging drop method. Crystals were obtained by microseeding (touching 3D crystal) and using mother liquor of 20% PEG 4000, 200 mM NaCl, 200 mM ammonium sulphate in 100 mM Na citrate buffer, pH 5.6. Before freezing, crystals were soaked in mother liquor with 20% ethyleneglycol.

            In spite of large and optically perfect crystals, the successful X-ray diffraction measurement required very high intensity of primary X-ray beam. The final measurement was performed at the source of synchrotron radiation ESRF in Grenoble at the beam line ID 29. Because of large unit cell (a = 140.1 Å, b = 205.7 Å, c = 140.5 Å, β = 102.3°, space group P21), the diffraction data was collected with small oscillation angle 0.1°. Total 1800 frames gave 28 millions of measurements up to resolution 1.9 Å. They were processed by special version of  the HKL package of programs [3] for viruses. All data processed up to 1.9 Å gave 579 289 unique reflections, Rlin = 8.5 % and completeness 95 %. The asymmetric unit contains one tetramer.

            The phase problem was solved by the program EPMR (molecular replacement by evolutionary search [4]) using the structure of β-galactosidase from Escherichia coli (PDB code 1DP0, resolution 1.7 Å) as a model. In spite of low homology, the global structure appears similar. The structure building and the refinement is under progress.


Acknowledgment. The research was supported by the Grant Agency of the Czech Republic (project 204/02/0843/A) and by the Academy of Sciences of the Czech Republic (project AVOZ4050913).



[1] P. Karasová, V. Spiwok, Š. Malá, B. Králová, N. J. Russell, Beta-galactosidase activity in psychrotrophic microorganisms and their potential use in food industry, Czech J. Food Sci. (2002) 43-47.


[2] Karasová-Lipovová P., Strnad H., Spiwok V., Malá Š., Králová B., Russell N. J., The cloning, purification and characterisation of a cold-active beta-galactosidase from the psychrotolerant Antarctic bacterium Arthrobacter sp C2-2,  Enzyme Microb. Tech. (2003) 836-844.


[3] Z. Otwinowski and W. Minor, " Processing of X-ray Diffraction Data Collected in Oscillation Mode ", Methods in Enzymology, Volume 276: Macromolecular Crystallography, part A (1997) 307-326, C.W. Carter, Jr. & R. M., Sweet, Eds., Academic Press (New York).


[4] Charles R. Kissinger, Daniel K. Gehlhaar, & Bradley A. Smith, Agouron Pharmaceuticals, Inc, EPMR - A program for crystallographic molecular replacement by evolutionary search. Version 2.5;