Crystallographic studies of a newly prepared histidinol–phosphate phosphatase Tt82 from Thermococcus onnurineus

Petra Havlickova1, Tatyana Prudnikova1,3, Jeroen R. Mesters2, Michal Kuty1,3,
Marc L. Pusey4 and Ivana Kuta Smatanova1,3

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

2Institute of Biochemistry, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany

3Academy of Sciences of the Czech Republic, Center for Nanobiology and Structural Biology IMB, Zamek 136, 373 33 Nove Hrady

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

Histidinol-phosphate phosphatase (HolPase, EC 3.1.3.15) catalyzes the dephosphorylation of histidinol-phosphate to histidinol, which is the eighth step in the histidine biosynthesis pathway [1]. Histidinol-phosphate phosphatases belong to the HAD superfamily, crystal structures from the HAD superfamily share a conserved α/β-domain classified as a hydrolase fold and usually contain an insertion subdomain [2]. Protein Tt82 from Thermococcus onnurineus is a 241-residues protein and according to the analysis of its primary structure it is considered to be a histidinol-phosphate phosphatase. Crystal screening was performed with screening kit Index-HR-144 (Hampton Research, USA) using the sitting drop vapor diffusion method at 295 K, applying an Oryx-6 crystallization robot (Douglas Instruments Hungerford, England). Optimization of preliminary crystallization conditions yielding crystals was performed manually with the sitting drop vapor diffusion method, varying the drop ratio and protein concentration. The 3D crystals suitable for X-ray diffraction measurement were grown in approximately 3 weeks from the precipitant composed of 0.2 M Magnesium chloride hexahydrate, 25% w/v Polyethylene glycol 3350, 0.1 M Tris pH 8.5. 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). Tt82 crystals belonged to space group P212121, with unit cell parameters a = 66.59, b = 117.47, c = 33.97 Å. 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 [3] and Phaser [4] resulted in failure. Therefore, heavy atom derivative crystals were grown by co-crystallization procedure with the final concentration in drop of 1 mM Manganese (II) chloride (the X-ray absorption edge is 6.5390 keV) in order to solve the crystal structure using the multiple wavelength anomalous diffraction method.

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The work was supported by GACR 17-24321S, GAJU 04-149/2016/P and ERDF No. CZ.02.1.01/0.0/0.0/15_003/0000441.