RESUMO
Uridine phosphorylase (UPh) catalyzes the phosphorolytic cleavage of the C-N glycosidic bond of uridine to ribose 1-phosphate and uracil in the pyrimidine-salvage pathway. The crystal structure of the Salmonella typhimurium uridine phosphorylase (StUPh) has been determined at 2.5 A resolution and refined to an R factor of 22.1% and an Rfree of 27.9%. The hexameric StUPh displays 32 point-group symmetry and utilizes both twofold and threefold non-crystallographic axes. A phosphate is bound at the active site and forms hydrogen bonds to Arg91, Arg30, Thr94 and Gly26 of one monomer and Arg48 of an adjacent monomer. The hexameric StUPh model reveals a close structural relationship to Escherichia coli uridine phosphorylase (EcUPh).
Assuntos
Salmonella typhimurium/enzimologia , Uridina Fosforilase/química , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/enzimologia , Ligação de Hidrogênio , Estrutura Molecular , Conformação ProteicaRESUMO
The structural udp gene encoding uridine phosphorylase (UPh) was cloned from the Salmonella typhimurium chromosome and overexpressed in Escherichia coli cells. S. typhimurium UPh (StUPh) was purified to apparent homogeneity and crystallized. The primary structure of StUPh has high homology to the UPh from E. coli, but the enzymes differ substantially in substrate specificity and sensitivity to the polarity of the medium. Single crystals of StUPh were grown using hanging-drop vapor diffusion with PEG 8000 as the precipitant. X-ray diffraction data were collected to 2.9 A resolution. Preliminary analysis of the diffraction data indicated that the crystal belonged to space group P6(1(5)), with unit-cell parameters a = 92.3, c = 267.5 A. The solvent content is 37.7% assuming the presence of one StUPh hexamer per asymmetric unit.
