Crystallization studies and preliminary X-ray crystallographic analysis of a newly prepared histidinol–phosphate phosphatase Tt82 from Thermococcus onnurineus

Petra Havlickova¹, Tatyana Prudnikova^1,2, Jeroen R. Mesters³, Michal Kuty^1,2, Marc L. Pusey⁴ and Ivana Kuta Smatanova^1,2

¹University of South Bohemia in Ceske Budejovice, Faculty of Science, Branisovska 1760, 370 05 Ceske Budejovice, Czech Republic

²Academy of Sciences of the Czech Republic, Center for Nanobiology and Structural Biology IMB, Zamek 136, 373 33 Nove Hrady, Czech Republic

³Institute of Biochemistry, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany

⁴iXpressGenes Inc.,601 Genome Way, Huntsville, AL 35810, USA

Dephosphorylation of histidinol–phosphate is the eight step in the histidine-biosynthetic pathway and is catalyzed by histidinol-phosphate phosphatase [1]. The main goal of this research was to optimize crystallization conditions and obtain suitable monocrystals of a newly prepared and until now uncharacterized protein Tt82 from Thermococcus onnurineus, which is considered to be a histidinol–phosphate phosphatase. Crystal screening was performed with crystallization kit Index-HR2-144 (Hampton Research, USA) using sitting drop vapor diffusion technique at 295 K. The most suitable crystals for diffraction experiment were grown from the precipitant solution containing 0.2 M magnesium chloride hexahydrate, 0.1 M Tris pH 8.5 and 25% w/v PEG 3350 (condition No. 85). Native data set was collected to the resolution of 1.6 Å at the BESSY-II synchrotron operated by Joint Berlin MX-Laboratory (Berlin-Adleshof, Germany). Although attempting to solve the crystal structure using molecular-replacement method with homologues (PDB codes 1qyi, 3pib, 3kbb, 2hdo, 3r3h, 2vvl, 3iru) as search models, all trials using MOLREP [2] and Phaser [3] resulted in failure. Therefore, heavy atom derivative crystals were grown from the precipitant solution No. 85 from Index-HR2-144 (Hampton Research, USA) adding 1 mM manganese (II) chloride and data set is about to be collected. Diffraction data will be used for further research, mainly for solving the structure by heavy atom derivatization method.

1. Brilli, M. & Fani, R. (2004). J. Mol. Evol. 58, 225-237.

2. Vagin, A. & Teplyakov, A. (1997). J. Appl. Cryst. 30(6): 1022-1025.

3. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., Read, R. J. (2007). J. Appl. Cryst. 40(4): 658-674.

The work was supported from GACR 17-24321S and ERDF No. CZ.02.1.01/0.0/0.0/15_003/0000441 .