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Acta Crystallographica Section F: Structural Biology and Crystallization Communications logoLink to Acta Crystallographica Section F: Structural Biology and Crystallization Communications
. 2009 May 22;65(Pt 6):601–603. doi: 10.1107/S1744309109016133

Crystallization and preliminary X-ray diffraction analysis of Salmonella typhimurium uridine phosphorylase complexed with 5-fluorouracil

A A Lashkov a, A G Gabdoulkhakov a, A A Shtil b, A M Mikhailov a,*
PMCID: PMC2688420  PMID: 19478441

Uridine phosphorylase from S. typhimurium was expressed and purified and cocrystallized with the drug 5-fluorouracil. The crystals diffracted X-rays to 2.2 Å resolution using synchrotron radiation.

Keywords: uridine phosphorylase, 5-fluorouracil, Salmonella typhimurium

Abstract

Uridine phosphorylase (UPh; EC 2.4.2.3) catalyzes the phosphorolytic cleavage of the N-glycosidic bond of uridine to form ribose 1-phosphate and uracil. This enzyme also activates pyrimidine-containing drugs, including 5-fluorouracil (5-FU). In order to better understand the mechanism of the enzyme–drug interaction, the complex of Salmonella typhimurium UPh with 5-FU was cocrystallized using the hanging-drop vapour-diffusion method at 294 K. X-ray diffraction data were collected to 2.2 Å resolution. Analysis of these data revealed that the crystal belonged to space group C2, with unit-cell parameters a = 158.26, b = 93.04, c = 149.87 Å, α = γ = 90, β = 90.65°. The solvent content was 45.85% assuming the presence of six hexameric molecules of the complex in the unit cell.

1. Introduction

5-Fluorouracil (5-FU) has been used in chemotherapeutic regimens in patients with gastrointestinal malignancies, including oesophageal, gastric, colon and pancreatic tumours, as well as breast cancer for several decades (Kemeny, 1987 ▶; Huang et al., 2007 ▶). This drug competes with uracil for interaction with thymidylate synthase. 5-FU interferes with DNA and RNA synthesis, thereby causing cell death. Furthermore, 5-halogen-containing derivatives of uracil are also used as antimicrobial agents. Synergism of 5-FU with antibacterial agents such as ceftriaxone, ceftazidime, cefotiam, piperacillin and netilmicin has been shown (Price et al., 1965 ▶; Gieringer et al., 1986 ▶; Yamashiro et al., 1986 ▶). However, toxicity towards haematopoietic cells, skin, heart and neurons can limit the therapeutic efficacy of 5-­FU and structurally similar drugs (Peters & van Groeningen, 1991 ▶). Uridine phosphorylase (UPh) catalyzes the phosphorolytic cleavage of the N-glycosidic bond in uridine to produce ribose 1-phosphate and uracil (Leer et al., 1977 ▶; Vita et al., 1986 ▶). This enzyme activates pyrimidine-based drugs, including 5-FU (Cao et al., 2002 ▶; Caradoc-Davies et al., 2004 ▶). Therefore, studies of the structure of UPh bound to 5-FU should be instrumental in future modifications aimed at the design of 5-FU derivatives with lower general toxicity and retained antitumour and antimicrobial potencies (Iigo et al., 1990 ▶; Temmink et al., 2006 ▶; Matsusaka et al., 2007 ▶).

2. Protein expression and purification

Salmonella typhimurium UPh (StUPh) was expressed in Escherichia coli and purified as reported elsewhere (Molchan et al., 1998 ▶; Dontsova et al., 2004 ▶). The Escherichia coli BL21 (DE3) strain was transformed with the recombinant plasmid and grown on solid LB agar for 12 h at 310 K. Protein synthesis was stimulated with 0.5 mM isopropyl β-d-1-thiogalactopyranoside. The biomass was sonicated, ammonium sulfate was added to precipitate the proteins and the pellets were dissolved in buffer (pH 7.2) containing 50 mM KH2PO4 and 0.5 mM β-mercaptoethanol. Further steps of StUPh purification were performed using chromatography on butyl Sepharose as a first step and Q-Sepharose as the final step. The enzymatic activity was 280 units per milligram of purified protein. The homogeneity of the purified StUph was 96% as determined by nondenaturing gel electrophoresis.

3. Crystallization of the StUph–5FU complex

Crystals of the complex of StUph with 5-FU (EBEWE Pharma, Austria) were obtained by cocrystallization (Fig. 1 ▶). Crystallization was performed on siliconized glass slides (Hampton Research, USA) in Linbro plates at 294 K using the hanging-drop vapour-diffusion method. The reservoir solution (0.5 ml) consisted of 0.34 ml 0.1 M Tris–maleate–NaOH buffer pH 5.5 and 0.16 ml 40%(w/v) polyethylene glycol 3350. The crystallization drop contained 2 µl StUph solution (11.3 mg ml−1) in 10 mM Tris–HCl buffer pH 7.3, 2 µl H2O, 1.3 µl reservoir solution, 2 µl 100 mM 5-FU and 0.3 µl 2-­propanol. Crystals of dimensions 0.07 × 0.3 × 0.5 mm were obtained after 1–2 weeks and were used for X-ray diffraction analysis.

Figure 1.

Figure 1

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Crystal of StUPh complexed with 5-FU.

4. X-ray analysis

The X-ray data set (Table 1 ▶) was collected upon irradiation of StUPh–5FU crystals under cryogenic conditions (100 K) on beamline 14.2 at BESSY, Berlin, Germany. The wavelength was 0.9184 Å. A CHESS CCD detector was used with an oscillation range Δϕ of 0.5° and a crystal-to-detector distance of 240 mm. Prior to freezing in liquid nitrogen, the crystals were transferred into cryoprotectant solution containing 100 mM Tris–maleate–NaOH buffer pH 5.5, 25%(w/v) polyethylene glycol 3350 and 20%(v/v) anhydrous glycerol. All data were processed and scaled using XDS (Kabsch, 1988 ▶).

Table 1. Statistics of X-ray data.

Values in parentheses are for the last resolution shell.

Wavelength (Å) 0.918
Temperature (K) 100
Oscillation (°) 0.5
Space group C2
Unit-cell parameters (Å, °) a = 158.26, b = 93.04, c = 149.87, α = γ = 90, β = 90.65
Molecules per unit cell 6
VM (Å3 Da−1) 2.27
Solvent content (%) 45.85
Resolution limits (Å) 30.0–2.2 (2.25–2.2)
Completeness (%) 90.2 (79.3)
No. of reflections 324446
No. of unique reflections 99573 (7124)
Robserved (%) 6.8 (55.6)
Rexpected (%) 7.0 (55.3)
〈I/σ(I)〉 12.67 (2.16)
Rmeas 0.08 (0.66)
Rmerge 0.017 (0.47)
Redundancy 3.26 (3.24)
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300 images were obtained during the collection of X-ray diffraction data. All data were indexed, merged and processed using the XDS program with XYCORR, INIT, COLSPOT, IDXREF, DEFPIX, XPLAN, INTEGRATE and CORRECT options. For a semi-automatic determination of the space group, the minimal value of the XDS ‘quality of fit’ function was used. The crystals of the StUPh–5-­FU complex belonged to space group C2. Detailed data statistics are presented in Table 1 ▶.

The structure was resolved by the molecular-replacement technique using the Phaser program with rigid-body refinement option (McCoy, 2007 ▶). X-ray diffraction data from 10 to 2.5 Å resolution were used in this step. The X-ray structure of monomer A only of ligand-free StUPh at 1.76 Å resolution (Timofeev et al., 2007 ▶; PDB code 2oxf) was utilized as a search model. Water molecules were removed from the model. Six full homohexamer molecules were found in the unit cell. The Matthews coefficient (Matthews, 1968 ▶) was 2.27 Å3 Da−1 and the solvent content was 45.85% (Table 1 ▶). Only one solution was evident, with an R factor of 37.64% and a correlation coefficient R corr of 76.95%.

To improve the phase model, one macrocycle of simulated annealing using the phenix.refine module of PHENIX (Adams et al., 2002 ▶) was performed in the temperature range 12 000–300 K with 50 K steps and resolution 10–2.2 Å. Before refinement, 5% of the observations were chosen at random and set aside for cross-valid­ation analysis and to monitor the various refinement strategies. Next, σA-weighted electron-density maps with coefficients (2|F o| − |F c|) and (|F o| − |F c|) were obtained using PHENIX. Using the (|F o| − |F c|) electron-density map in the Coot program (Emsley & Cowtan, 2004 ▶), we identified 5-FU molecules and water molecules localized in the close vicinity of the 5-FU molecules. After two cycles of restrained refinement in REFMAC (Murshudov et al., 1997 ▶) and the synthesis of σA-weighted (2|F o| − |F c|) and (|F o| − |F c|) electron-density maps the R factor was 26.42% and R free was 29.24%.

One uracil-binding site in StUph complexed with 5-FU and water is shown in Fig. 2 ▶. The cartoon representation was generated with PyMOL (DeLano, 2008 ▶). The drug and water bind to the following atoms of the amino-acid residues in the uracil-binding site: Arg223 NH1–water (2.85 Å), water–5-FU O4 (2.88 Å), Arg168 NH1–5-FU O4 (3.00 Å), Gln166 OE1–5-FU N3 (2.70 Å), Gln166 NE2–5-FU O2 (2.89 Å). Refinement of the structure is currently in progress and will be published elsewhere.

Figure 2.

Figure 2

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Preliminary structure of the uracil-binding site in the StUPh–5-FU complex.

Acknowledgments

This work was supported partially by RFBR and Kaluga region administration (grant No. 09-02-97519).

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