Purification, crystallization and preliminary X-ray analysis of uridine phosphorylase fromSalmonella typhimurium

Abstract

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.

Preliminary investigation of the three-dimensional structure ofSalmonella typhimuriumuridine phosphorylase in the crystalline state

“…StUPh was overexpressed in E. coli and purified by hydrophobic chromatography as described previously (Dontsova et al, 2004;Mikhailov et al, 1992;Molchan et al, 1998). Fractions containing pure StUPh were pooled and dialyzed into buffer containing 10 mM Tris-HCl pH 7.3, 0.4% NaN 3 and concentrated to 20 mg ml À1 .…”

X-ray structure ofSalmonella typhimuriumuridine phosphorylase complexed with 5-fluorouracil and molecular modelling of the complex of 5-fluorouracil with uridine phosphorylase fromVibrio cholerae

“…The procedures for the isolation and purification of StUPh have been described previously (Dontsova et al, 2004;Molchan et al, 1998). Briefly, DNA isolated from S. typhimurium was cloned into pBluescript II SK vector (Zolotukhina et al, 2003).…”

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

“…The supernatant was incubated at 277 K for 3 h; proteins were then precipitated with polyethylene glycol (PEG) and resuspended in buffer containing 10 mM KH 2 PO 4 pH 7.2 and 0.5 mM -mercaptoethanol. Further purification of StUPh was performed using butyl-Sepharose chromatography and Q-Sepharose chromatography (Dontsova et al, 2004). The homogeneity of the native StUPh was determined by electrophoresis in a 7.5% polyacrylamide gel under nondenaturing conditions.…”

“…Unlike StUPh and the E. coli orthologue EcUPh, the quaternary structure of HUPhI is represented by a homodimer (Roosild et al, 2009). However, in all eukaryotic and prokaryotic UPhs studied to date the homodimer is the major structural and functional unit (Burling et al, 2003;Caradoc-Davies et al, 2004;Dontsova et al, 2004;Roosild et al, 2009).…”

“…The key residues that interact with uracil in StUPh are Gln166B, Arg168B and Arg223B. These residues are conserved in bacterial phosphorylases and are important for recognition of the pyrimidine ring in substrates (Burling et al, 2003;Dontsova et al, 2004;Morgunova et al, 1995). In the uracil binding site, O4 of the uracil moiety of ANU and the respective atom in BAU form hydrogen bonds to the same atoms (Bu et al, 2005).…”