Structure changes evoked by binding of ligands into the active site of enzyme β-galactosidase from Arthrobactersp. C2-2

Andrea Štěpánková1, 2, Tereza Skálová2, Jan Dohnálek2, Jarmila Dušková2, Petr Kolenko1, 2, Jindřich Hašek2,  Vojtěch Spiwok3, Petra Lipovová3

 

1Department of Solid State Physics, FNSPE, CTU, Trojanova 13, 120 00, Prague 2, Czech Republic

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

3Dept. of Biochemistry, ICT, Technická 5, 166 28, Prague 6, Czech Republic

stepanko@imc.cas.cz

 

The structures of three complexes of enzyme β-galactosidase from an Antarctic bacterium Arthrobacter sp. C2-2 are presented. Three of the obtained crystals with dimensions 100 – 400 µm were soaked in different ligands (D-galactose, D-galactonolactone and IPTG (Isopropyl β-D-1-thiogalactopyranoside) ) and used for X-ray diffraction data collection.

IPTG is an inhibitor for this enzyme, diffraction data were collected on an in-house source of X-ray radiation using rotating anode. The resolution limit is 3.3 Å. The data for complexes with D-galactonolactone and D-galactose were collected in ESRF in Grenoble using synchrotron radiation and the structures were determined at resolutions of 2.2 and 2.5 Å. D-galactonolactone is an inhibitor for this enzyme and the D-galactose is a product of the catalyzed reaction. The data were processed using HKL2000. All the crystals belong to monoclinic space group P21, the unit cells parameters are very similar but packing of the hexamers in the crystals differs.

The enzyme β-galactosidase (EC 3.2.1.23) belongs to the enzyme class called glycosylases which catalyze hydrolysis of the terminal β-D-galactosyl of β-D-galactosides and it is able to catalyze trans-glycosylation. It is attractive for research and industry because of its wide range of biotechnological applications (to reduce the energy costs, to treat lactose intolerance, to prevent crystallization in sweet products, to increase its sweetening power, to simplify  fermentation during production of soured milk products, to modify the freezing point of ice creams, etc.). β-galactosidases are distributed in numerous microorganism, plants and animal tissues. Enzymes that exhibit β-galactosidase activity are derided into into four distinct GHs, GH-1, GH-2, GH-35 and GH-42. β-galactosidase from Escherichia coli, which belongs to GH-2, is one of the most studied and commonly used β-galactosidases. The three-dimensional structure of β-galactosidase1 from Escherichia coli has been determined.

This project is focused on β-galactosidase from an Antarctic bacterium Arthrobacter sp. C2-2. Psychrotrophic bacterium Arthrobacter sp. C2-2 was isolated in the Antarctic area as  part of an environmental study. This psychrotrophic bacterium is able to exist at low temperature (which is typical for Arctic and Antarctic regions, and for mountains and deep oceans too).

The structure of native enzyme β-galactosidase2 from Arthrobacter sp. C2-2 has been determined. There are several structure features in β-galactosidase from Arthrobacter sp. C2-2. The biggest one is that this enzyme forms hexamer with molecular weight of 660 kDa.  These hexamers were indicated in solution and in asymmetric unit of crystal in contrast to β-galactosidase from E. coli which forms homotetramers. Each monomer consists of five domains and contains 1023 residues and has an active site located in the TIM barrel domain - between the pair of catalytic residues Glu442 and Glu521. There are two distinct binding modes for the galactosyl group of ligands - shallow and deep. Each binding mode has specific hydrogen bonds between enzyme and ligands. In the cold adapted enzyme, the residue Trp552 is responsible for binding in the deep binding mode and the residue Cys999 for binding in the shallow binding mode.

IPTG is bound in the shallow binding mode. IPTG is located on the top of the active site and in contact with Cys999. IPTG is bound in the very similar position as IPTG in the structure of the mesophilic β-galactosidase.

The molecules of D-galactonolactone and D-galactose were found in the deep binding mode. In comparison with the shallow binding mode, the galactosyl moiety is rotated by about 90° and shifted deeper into the active site to rest on Trp552.

The molecule of D-galactose was found in a chair conformation in each monomer. Its oxygen atom 1 is in β-anomer configuration. The galactose 6-hydroxyl binds directly to the sodium ion. The binding is not accompanied by any conformational change of the enzyme as opposed to the complex with D-galactonolactone. There is only a small difference between the molecule of D-galactose and D-galactonolactone: D-galactonolactone has the double bond between carbon C1 and oxygen O1 atoms. This small difference causes a large structural change in the case of the complex with D-galactonolactone.

The molecule of D-galactonolactone was found in the active site of each monomer. The 6-hydroxyl binds directly to the sodium ion in all three structures. The binding is accompanied by an enzyme conformational change - Phe585 is rotated and the side chain of His335 moved up closer to the active site.

1.       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.

2.        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.

3.        Otwinovsky, Z., Minor, W., (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol., 276, 307-326.

 

Acknowledgements:

       This work was supported by the Czech Science Foundation (project  305/07/1073) and by the IGS CVUT (project CTU0803914).