Crystallization of Carbohydrate oxidase from Microdochium nivale

J. Dušková¹, J. Dohnálek^{1, 2}, T. Skálová¹, L. H. Østergaard³, C. C. Fuglsang³, P. Kolenko¹, A. Štěpánková¹ and J. Hašek

<aff><oid id="1">Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, Praha 6, <cny>Czech Republic</cny></aff>,<aff><oid id="2">Institute of Physics, Academy of Sciences of the Czech Republic, Cukrovarnická 10, Praha 6, Czech Republic, <cny>Czech Republic</cny></aff>, and <aff><oid id="3">Novozymes A/S, Brudelysvej 26, DK-2880 Bagsvaerd, <cny>Denmark</cny></aff></aug>. E-mail: duskova@imc.cas.cz

Carbohydrate oxidase from Microdochium nivale containing 475 amino acids belongs to a family of flavin- or Cu-containing enzymes whose function consists in catalytic oxidation of the primary alcohol in various oligosacharides. The product of the reaction is the corresponding saccharide acid and the process is accompanied by reduction of molecular oxygen to hydrogen peroxide, which has large potential industrial utilization.

Hampton Research Crystal Screens and Index solutions were used for the initial screening and crystallization experiments were performed using vapour diffusion and micro-batch methods at 290 K and 298 K. Morphology of the crystals varied depending on crystallization conditions and crystals featured varying stability.

A hexagon-shaped crystal grown with Jeffamine ED2001 as precipitant was measured at beamline BM14, ESRF in Grenoble using the MARMosaic 225 detector. The data were collected up to 2.7 Å resolution, however, a rapid intensity fall-off occurred beyond 3.5 Å. The space group was assigned as P6₂22, with unit cell parameters a = b = 55.7 Å, c = 610.4 Å. The crystallographic symmetry was verified with program Pointless [1]. Analysis of the intensity statistics clearly showed the presence of merohedral twinning. This was confirmed by the web-based service for analysis of twinning http://nihserver.mbi.ucla.edu/Twinning which indicated merohedral twinning with a twin factor of 43% [2].

Extensive molecular replacement trials with a model of 41% sequence identity (PDB code 1zr6) [3] were performed in MOLREP [4] and AMORE [5], however, without satisfactory results. Further experiments to produce well diffracting crystals are necessary.

1. Collaborative Computational Project, Number 4, Acta Cryst., D50, (1994),760-763.

2. Yeates, T. O., Methods Enzymol., 276, (1997), 344-358.

3. Huang, C, Lai, W, Lee, M. H., Chen, C. J., Vasella, A., Tsai, Y. C., & Liaw, S. H., J. Biol. Chem,. 280, (2005), 38831 - 38838.

4. Vagin A. & Teplyakov, A., J. Appl. Cryst. 30, (1997), 1022-1025.

5. Navaza, J., Acta Cryst., A50, (1994), 157-163.

Acknowledgement:

This work was supported by GA AV ČR, project IAA500500701 and by GA ČR, project 305/07/1073.