Expression, purification, crystallization and preliminary X-ray structure analysis ofVibrio choleraeuridine phosphorylase in complex with thymidine

Abstract

A high-resolution structure of the complex ofVibrio choleraeuridine phosphorylase (VchUPh) with its physiological ligand thymidine is important in order to determine the mechanism of the substrate specificity of the enzyme and for the rational design of pharmacological modulators. Here, the expression and purification ofVchUPh and the crystallization of its complex with thymidine are reported. Conditions for crystallization were determined with an automated Cartesian Dispensing System using The Classics, MbClass and MbClass II Suites crystallization kits. Crystals of theVchUPh–thymidine complex (of dimensions ∼200–350 µm) were grown by the sitting-drop vapour-diffusion method in ∼7 d at 291 K. The crystallization solution consisted of 1.5 µlVchUPh (15 mg ml−1), 1 µl 0.1 Mthymidine and 1.5 µl reservoir solution [15%(w/v) PEG 4000, 0.2 MMgCl2.6H2O in 0.1 MTris–HCl pH 8.5]. The crystals diffracted to 2.12 Å resolution and belonged to space groupP21(No. 4), with unit-cell parametersa = 91.80,b= 95.91,c= 91.89 Å, β = 119.96°. The Matthews coefficient was calculated as 2.18 Å3 Da−1; the corresponding solvent content was 43.74%.