RESUMO
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 â¼200-350â µm) were grown by the sitting-drop vapour-diffusion method in â¼7â d at 291â K. The crystallization solution consisted of 1.5â µl VchUPh (15â mgâ ml(-1)), 1â µl 0.1â M thymidine and 1.5â µl reservoir solution [15%(w/v) PEG 4000, 0.2â M MgCl(2).6H2O in 0.1â M Tris-HCl pH 8.5]. The crystals diffracted to 2.12â Å resolution and belonged to space group P2(1) (No. 4), with unit-cell parameters a=91.80, b=95.91, c=91.89â Å, ß=119.96°. The Matthews coefficient was calculated as 2.18â Å3â Da(-1); the corresponding solvent content was 43.74%.
Assuntos
Proteínas de Bactérias/química , Timidina/química , Uridina Fosforilase/química , Vibrio cholerae/enzimologia , Motivos de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Modelos Moleculares , Uridina Fosforilase/isolamento & purificaçãoRESUMO
Uridine phosphorylase catalyzes the phosphorolysis of ribonucleosides, with the nitrogenous base and ribose 1-phosphate as products. Additionally, it catalyzes the reverse reaction of the synthesis of ribonucleosides from ribose 1-phosphate and a nitrogenous base. However, the enzyme does not catalyze the synthesis of nucleosides when the substrate is a nitrogenous base substituted at the 6-position, such as 6-methyluracil (6-MU). In order to explain this fact, it is essential to investigate the three-dimensional structure of the complex of 6-MU with uridine phosphorylase. 6-MU is a pharmaceutical agent that improves tissue nutrition and enhances cell regeneration by normalization of nucleotide exchange in humans. 6-MU is used for the treatment of diseases of the gastrointestinal tract, including infectious diseases. Here, procedures to obtain the uridine phosphorylase from the pathogenic bacterium Vibrio cholerae (VchUPh), purification of this enzyme, crystallization of the complex of VchUPh with 6-MU, and X-ray data collection and preliminary X-ray analysis of the VchUPh-6-MU complex at atomic resolution are reported.