ABSTRACT
Highly specific thymidine phosphorylases catalyze the phosphorolytic cleavage of thymidine, with the help of a phosphate ion, resulting in thymine and 2-deoxy-α-D-ribose 1-phosphate. Thymidine phosphorylases do not catalyze the phosphorolysis of uridine, in contrast to nonspecific pyrimidine nucleoside phosphorylases and uridine phosphorylases. Understanding the mechanism of substrate specificity on the basis of the nucleoside is essential to support rational drug-discovery investigations of new antitumour and anti-infective drugs which are metabolized by thymidine phosphorylases. For this reason, X-ray structures of the thymidine phosphorylase from Salmonella typhimurium were solved and refined: the unliganded structure at 2.05 Å resolution (PDB entry 4xr5), the structure of the complex with thymidine at 2.55 Å resolution (PDB entry 4yek) and that of the complex with uridine at 2.43 Å resolution (PDB entry 4yyy). The various structural features of the enzyme which might be responsible for the specificity for thymidine and not for uridine were identified. The presence of the 2'-hydroxyl group in uridine results in a different position of the uridine furanose moiety compared with that of thymidine. This feature may be the key element of the substrate specificity. The specificity might also be associated with the opening/closure mechanism of the two-domain subunit structure of the enzyme.
Subject(s)
Bacterial Proteins/chemistry , Salmonella typhimurium/enzymology , Thymidine Phosphorylase/chemistry , Thymine Nucleotides/chemistry , Uridine/chemistry , Amino Acid Sequence , Catalytic Domain , Crystallization , Crystallography, X-Ray , Ligands , Protein Binding , Substrate SpecificityABSTRACT
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.
Subject(s)
Uracil/analogs & derivatives , Uridine Phosphorylase/chemistry , Vibrio cholerae/enzymology , Binding Sites , Biocatalysis , Crystallization , Crystallography, X-Ray , Models, Molecular , Uracil/chemistryABSTRACT
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%.
Subject(s)
Bacterial Proteins/chemistry , Thymidine/chemistry , Uridine Phosphorylase/chemistry , Vibrio cholerae/enzymology , Amino Acid Motifs , Bacterial Proteins/isolation & purification , Catalytic Domain , Crystallization , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Models, Molecular , Uridine Phosphorylase/isolation & purificationABSTRACT
Uridine phosphorylase (UPh), which is a key enzyme in the reutilization pathway of pyrimidine nucleoside metabolism, is a validated target for the treatment of infectious diseases and cancer. A detailed analysis of the interactions of UPh with the therapeutic ligand 5-fluorouracil (5-FUra) is important for the rational design of pharmacological inhibitors of these enzymes in prokaryotes and eukaryotes. Expanding on the preliminary analysis of the spatial organization of the active centre of UPh from the pathogenic bacterium Salmonella typhimurium (StUPh) in complex with 5-FUra [Lashkov et al. (2009), Acta Cryst. F65, 601-603], the X-ray structure of the StUPh-5-FUra complex was analysed at atomic resolution and an in silico model of the complex formed by the drug with UPh from Vibrio cholerae (VchUPh) was generated. These results should be considered in the design of selective inhibitors of UPhs from various species.
Subject(s)
Fluorouracil/pharmacology , Salmonella typhimurium/enzymology , Uridine Phosphorylase/chemistry , Vibrio cholerae/enzymology , Catalysis , Catalytic Domain , Cluster Analysis , Enzyme Inhibitors/pharmacology , Ligands , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , SolventsABSTRACT
The ligninolytic enzymes of the basidiomycetes play a key role in the global carbon cycle. A characteristic property of these enzymes is their broad substrate specificity, which has led to their use in various biotechnologies, thus stimulating research into the three-dimensional structures of ligninolytic enzymes. This paper presents the purification, crystallization and preliminary X-ray analysis of the laccase from the ligninolytic basidiomycete Ganoderma lucidum.
Subject(s)
Laccase/chemistry , Reishi/enzymology , Crystallization , Crystallography, X-Ray , Laccase/isolation & purification , Models, Molecular , Protein Structure, TertiaryABSTRACT
Uridine nucleoside phosphorylase is an important drug target for the development of anti-infective and antitumour agents. The X-ray crystal structure of Salmonella typhimurium uridine nucleoside phosphorylase (StUPh) complexed with its inhibitor 2,2'-anhydrouridine, phosphate and potassium ions has been solved and refined at 1.86 A resolution (R(cryst) = 17.6%, R(free) = 20.6%). The complex of human uridine phosphorylase I (HUPhI) with 2,2'-anhydrouridine was modelled using a computational approach. The model allowed the identification of atomic groups in 2,2'-anhydrouridine that might improve the interaction of future inhibitors with StUPh and HUPhI.
Subject(s)
Bacterial Proteins/chemistry , Macromolecular Substances/chemistry , Salmonella typhimurium/enzymology , Uridine Phosphorylase/chemistry , Uridine/chemistry , Bacterial Proteins/metabolism , Crystallization , Crystallography, X-Ray , Humans , Ions/chemistry , Phosphates/chemistry , Potassium/chemistry , Protein Conformation , Uridine/analogs & derivatives , Uridine/pharmacology , Uridine Phosphorylase/metabolismABSTRACT
Uridine phosphorylase (UPh; EC 2.4.2.3) is a member of the pyrimidine nucleoside phosphorylase family of enzymes which catalyzes the phosphorolytic cleavage of the C-N glycoside bond of uridine, with the formation of ribose 1-phosphate and uracil. This enzyme has been shown to be important in the activation and catabolism of fluoropyrimidines. Modulation of its enzymatic activity may affect the therapeutic efficacy of chemotherapeutic agents. The structural investigation of the bacterial uridine phosphorylases, both unliganded and complexed with substrate/product analogues and inhibitors, may help in understanding the catalytic mechanism of the phosphorolytic cleavage of uridine. Salmonella typhimurium uridine phosphorylase has been crystallized with 2,2'-anhydrouridine. X-ray diffraction data were collected to 2.15 A. Preliminary analysis of the diffraction data indicates that the crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 88.52, b = 123.98, c = 133.52 A. The solvent content is 45.51%, assuming the presence of one hexamer molecule per asymmetric unit.