Crystallization and X-ray diffraction of an ectoine hydroxylase from Bacillus pseudofirmus OF4

To study crystallization and X-Ray diffraction of ectoine hydroxylase from Bacillus pseudofirmus OF4.

We cloned the gene BpectD from B. pseudofirmus OF4 with a His6 tag and overexpressed it in E. coli BL21 (DE3). Then, we purified protein BpEctD by Ni2 -chelating affinity and size-exclusion chromatography. After that, crystals were grown by the sitting-drop vapour-diffusion method at 289 K and diffracted at 100K using an in-house X-ray source.

Protein BpEctD was expressed and purified successfully. We obtained well diffracting crystals of about 360 μm x 240 μm x 60 μm in size using a solution consisting of 0.2 mol/L magnesium chloride hexahydrate, 0.1 mol/L bis-tris pH6. 5, 25% (W/V) polyethylene glycol 3,350 at a protein concentration of 6. 5 mg/mL, and collected X-ray diffraction data to 2.40 Å resolution in the anorthic space group P1, with unit-cell parameters a = 45.18Å, b = 58.87Å, c = 68.81 Å, α = 77.48 degrees, β = 86.03 degrees, γ = 66.97 degrees. The asymmetric unit contains two molecules of BpEctD with a Mattews coefficient of about 2.44 Å3/Da and a solvent content of 49.53%.

According to the X-ray diffraction data, the three-dimensional structure of BpEctD from B. pseudofirmus OF4 soon will be analyzed, and it will provide insights into the biochemical properties of ectoine hydroxylase.