Structure of b-galactosidase complexes

J. Dohnálek¹, T. Skálová¹, J. Dušková¹, H. Petroková¹, J. Hašek¹, P. Lipovová², V. Spiwok², H. Strnad³, B. Králová²

¹Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, 16206 Praha 6, Czech Republic

²Institute of Chemical Technology, Technická 5, 16628 Praha 6, Czech Republic

³Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 16637, Praha 6, Czech Republic

dohnalek@imc.cas.cz

The unliganded structure of b-galactosidase from the soil bacterium Arthrobacter sp. C2-2 isolated in Antarctica (GH family 2) revealed compact hexameric organization of the enzyme [1-2]. It is the first structure of a cold-active b-galactosidase. The structure was determined by single crystal X-ray crystallography with use of synchrotron radiation at the ESRF beamline ID29. The diffraction data were recorded in 1,800 oscillation images at 0.1º slicing and the scaled set of intensities contained 577,572 reflections in space group P2₁ (a = 140.1 Å, b = 205.7 Å, c = 140.5 Å, b = 102.3º). Six monomers of the enzyme are arranged with approximate 32 point symmetry into a sphere-like object and the individual active sites face the internal cavity. The cavity is connected with outer environment mainly by three different types of channels. The hexameric form is present in solution and is assumed to be the relevant biological oligomerization state. Therefore, ligands, substrates, ions and products interacting with the enzyme in the vicinity of the active site must enter and leave through the major openings. E.coli b-galactosidase from the same glycoside hydrolase family (a mesophilic counterpart) was extensively studied as for its oligomerization state (tetramer), activity, enzymatic mechanism and the a-complementation phenomenon [3]. Conclusions based on the new structure bring the two enzymes into contrast and raise questions regarding the hexamer’s function.

Diffraction data for several different complexes of the cold-active enzyme with various types of ligands were collected and processed, some of them with use of synchrotron radiation. It was confirmed that ligands must enter the internal cavity of the complex and thereby access the active sites of the hexamer.

1. H. Petroková, E. Vondráčková, T. Skálová, J. Dohnálek, P. Lipovová, V. Spiwok, H. Strnad, B. Králová, J. Hašek, Collect. Czech. Chem. Commun., 70, (2005), 124.

2. T. Skálová, J. Dohnálek, V. Spiwok, P. Lipovová, E. Vondráčková, H. Petroková, J. Dušková, H. Strnad, B. Králová, J. Hašek, J. Mol. Biol., 353, (2005), 282.

3. D.H. Juers, T.D. Heightman, A. Vasella, J.D. McCarter, L. Mackenzie, Biochemistry, 40, 14781.

Acknowledgements

This project was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (KJB500500512).