Galactonolactone inhibiting the active site of β-galactosidase from Arthrobacter sp. C2-2.

Crystal structure at 2.2 Å resolution

Andrea Štěpánková^1,2, Tereza Skálová², Jan Dohnálek², Jindřich Hašek², Petra Lipovová³

¹ Dept. of Solid State Physics, FNSPE, CTU, Trojanova 13, 120 00, Prague 2, stepanko@imc.cas.cz

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

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

Keywords: β-galactosidase, cold-active enzyme, X-ray structure.

The three-dimensional structure of enzyme beta-galactosidase from an Antarctic bacterium Arthrobacter sp. C2-2 with bound inhibitor has been determined at a resolution of 2.2 Å.

The enzyme β-galactosidase (EC 3.2.1.23) belongs to the enzyme class called glycosylases which catalyze the hydrolysis of the terminal beta-D-galactosyl of beta-D-galactosides. It is attractive for research and industry because of its wide range of biotechnological applications (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.).

The psychrotrophic bacterium Arthrobacter sp. C2-2 was isolated in the Antarctic area and this enzyme is active at low temperature, which is interesting property for food processing applications. Unlike, more known β-galactosidase from Escherichia coli, which form tetramers, the β-galactosidase from Arthrobacter sp. C2-2 forms hexamers with molecular weight of 660 kDa. Each monomer consists of five domains and contains 1023 residues. The active site is localized in the TIM barrel domain in the center of each monomer. The active site contains the pair of catalytic residues Glu442 and Glu521. The molecule of galactonolactone was found locked in the deep binding mode near the catalytic residues Glu442 and Glu521. Undoubtedly, the position of inhibitor closely simulates the transition state of galactose before the second step of enzymatic reaction (i.e. the release of product or the transglycosylation reaction).

X-ray diffraction data were collected at the source of synchrotron radiation ESRF in Grenoble. The data were processed using HKL-2000. The crystal belongs to space group P2₁ with unit cell parameters a = 140.3 Å, b = 205.5 Å, c = 140.5 Å, α = γ =90°, β = 102.5°. The structure is refined by REFMAC.

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

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

Murshudov, G.N., Vagin, A. A., Dodson, E. J. (1997). Refinement of macromolecular structures by the Maximum-Likelihood method. Acta Cryst. D53, 240-255.

This work was supported by the Grant Agency of the Czech Republic (project 204/02/0843/A), by the Grant Agency of the Academy of sciences of the Czech Republic (project KJB500500512) and by the Academy of Sciences of the Czech Republic (project AVOZ4050913).