Complexes of β-galactosidase with ligands


Andrea Štěpánková1, 2, Tereza Skálová1, Jan Dohnálek1, Jarmila Dušková1, Jindřich Hašek1, Petra Lipovová3, Tomáš Koval4


1 Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovského nám. 2, 162 00, Prague 6, Czech Republik

2 Dept. of Solid State Physics, FNSPE, CTU, Trojanova 13, 120 00, Prague 2, Czech Republic

3  Dept. of Biochemistry, ICT, Technická 5, 166 28, Prague 6, Czech Republic
4 Institute of Physics, Academy of Sciences of the Czech Republic, Cukrovarnická 10, Praha 6, Czech Republic


The three-dimensional structures of enzyme β-galactosidase1 from an Antarctic bacterium Arthrobacter sp. C2-2 with bound ligands have been determined at resolutions 2.2 Å, 2.5 Å and 3.3 Å.

Three of the obtained crystals with dimensions 100 – 400 µm were soaked in different ligands and used for X-ray diffraction data collection. D-galactonolactone and isopropyl β-D-thiogalactopyranoside (IPTG) are inhibitors and D-galactose is a product of the catalyzed reaction.

X-ray diffraction data were collected at the beam-line ID14.1 of the source of synchrotron radiation ESRF in Grenoble and on an in-house rotating anode diffractometer. The data were processed using HKL2000. All the three crystals belong to the same space group P21 but packing of hexamers in the crystals differs.

Binding of the reaction product confirms behavior similar to the mesophilic enzyme from E.coli whereas D-galactonolactone interaction with the enzyme brings up some interesting structural changes and related questions. The molecule of inhibitor D-galactonolactone was bound in the active site as  d-galactono-1,5-lactone, which is its the least probable isomer3, despite the fact that the crystal was soaked in an isomer  d-(−)-galactono-1,4-lactone. This  δ isomer creates specific bonds to Glu442 and ion Na+.

The enzyme β-galactosidase (EC belongs to the enzyme class called glycosylases which catalyze hydrolysis of the terminal β-D-galactosyl moiety of β-D-galactosides. It is attractive for research and industry because of its wide range of biotechnological applications (treatment for lactose intolerance, prevention of crystallization in sweet products and increase of sweetening power of saccharides, simpler fermentation during production of soured milk products, etc.). Unlike the more studied β-galactosidase from Escherichia coli2, which forms tetramers, the enzyme from Arthrobacter sp. C2-2 forms hexamers with a molecular weight of 660 kDa. Each monomer consists of five domains and contains 1023 residues.



1.       Skálová, T., Dohnálek, J., Spiwok, V., Lipovová, P., Vondráčková, E., Petroková, H., Dušková, J., Strnad, H., Králová, B., Hašek, J. (2005). Cold-active β-galactosidase from Arthrobacter sp. C2-2 forms compact 660 kDa hexamers: Crystal structure at 1.9 Å resolution. J. Mol. Biol., 353, 282-294.

2.       Jacobson, R. H., Zhang, X.-J., DuBose, R. F., Matthews, B. W. (1994). Three-dimensional structure of β-galactosidase from E. coli. Nature, 369, 761-766.

3.       Bierenstiel M., Schalf M.: Eur. J. Org. Chem., 2004/7,1474 (2004).



This work was supported by GA ČR, project 305/07/1073 and project 310/09/1407.