Isolation, crystallization and preliminary crystallographic analysis ofSalmonella typhimuriumuridine phosphorylase crystallized with 2,2′-anhydrouridine

Abstract

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 0 -anhydrouridine. X-ray diffraction data were collected to 2.15 Å . 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 Å . The solvent content is 45.51%, assuming the presence of one hexamer molecule per asymmetric unit.

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

“…The activity of UPh is known to be vital in some pathogenic bacteria and protozoa (Jimé nez et al, 1989;Lee et al, 1988). In previous publications, we have reported structures of UPh from Salmonella typhimurium with physiological ligands and pharmacological inhibitors (Dontsova et al, 2004(Dontsova et al, , 2005Timofeev et al, 2007;Lashkov et al, 2009Lashkov et al, , 2010Lashkov et al, , 2012. However, the diffraction resolution of complexes of bacterial UPh with thymidine does not allow conclusions to be drawn regarding the details of enzyme-ligand binding.…”

The X-ray structure ofSalmonella typhimuriumuridine nucleoside phosphorylase complexed with 2,2′-anhydrouridine, phosphate and potassium ions at 1.86 Å resolution

“…Crystals were grown by the hanging-drop vapour-diffusion method (Timofeev et al, 2007). The complex of StUPh with ANU, phosphate and potassium ions was obtained by cocrystallization.…”