title: Expression, purification, crystallization and preliminary X-ray structure analysis of Vibrio cholerae uridine phosphorylase in complex with thymidine.
abstract: A high-resolution structure of the complex of Vibrio cholerae uridine 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 of VchUPh 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 the VchUPh-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 µl VchUPh (15 mg ml(-1)), 1 µl 0.1 M thymidine and 1.5 µl reservoir solution [15%(w/v) PEG 4000, 0.2 M MgCl(2).6H2O in 0.1 M Tris-HCl pH 8.5]. The crystals diffracted to 2.12 Å resolution and belonged to space group P2(1) (No. 4), with unit-cell parameters a=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%.
authors.affiliation: A. V. Shubnikov Institute of Crystallography, Russian Academy of Sciences, Leninsky Prospect 59, Moscow 119333, Russian Federation.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Lashkov
authors.forename: Azat G
authors.initials: AG
authors.lastname: Gabdulkhakov
authors.forename: Igor I
authors.initials: II
authors.lastname: Prokofev
authors.forename: Tatyana A
authors.initials: TA
authors.lastname: Seregina
authors.forename: Sergey E
authors.initials: SE
authors.lastname: Sotnichenko
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Lyashenko
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Shtil
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Mironov
authors.forename: Christian
authors.initials: C
authors.lastname: Betzel
authors.forename: Al'bert M
authors.initials: AM
authors.lastname: Mikhailov
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D020134:Catalytic Domain; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008958:Models, Molecular; D013936:Thymidine / Q000737:chemistry*; D014543:Uridine Phosphorylase / Q000737:chemistry* / Q000302:isolation & purification; D014734:Vibrio cholerae / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D014543:Uridine Phosphorylase; D013936:Thymidine