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2.4.2.3: uridine phosphorylase

This is an abbreviated version!
For detailed information about uridine phosphorylase, go to the full flat file.

Word Map on EC 2.4.2.3

Reaction

uridine
+
phosphate
=
Uracil
 +
alpha-D-ribose 1-phosphate

Synonyms

apUP, EC 2.4.2.23, L-UrdPase, More, PcUP1, PcUP2, phosphorylase, uridine, pynpase, pyrimidine nucleoside phosphorylase, pyrimidine phosphorylase, pyrimidine/purine nucleoside phosphorylase, StUPh, udp, UDRPase , UP type 1, UP1, UP2, uPA, UPase, UPase-2, UPb, UPH, UPP1, UPP2, UrdPase, uridine phosphorylase, uridine phosphorylase 1, uridine phosphorylase-1, uridine phosphorylase-2, uridine:orthophosphate alpha-D-ribosyltransferase, VchUPh

ECTree

     2 Transferases
         2.4 Glycosyltransferases
             2.4.2 Pentosyltransferases
                2.4.2.3 uridine phosphorylase

Crystallization

Crystallization on EC 2.4.2.3 - uridine phosphorylase

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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method
-
hanging-drop vapour-diffusion method, crystals of thymidine phosphate complex, 2'-deoxyuridine complex, 5-fluorouracil ribose 1-phosphate complex and native enzyme form
hanging-drop vapour-diffusion method, structure at 2.0 A resolution
hanging-drop vapour-diffusion method, structures of 5-benzylacyclouridine, 5-phenylthioacyclouridine, 5-phenylselenylacyclouridine, 5-m-benzyloxybenzyl acyclouridine and 5-m-benzyloxybenzyl barbituric acid acyclonucleoside bound to the active site of the enzyme, resolutions ranging from 1.95 to 2.3 A
free enzyme and enzyme in complex with inhibitor 5-benzylacyclouridine, 3 mg/ml protein in absence or presence of 1 mM inhibitor from 17% PEG 3350, 100 mM Bis-Tris buffer, pH 5.5, 300 mM KCl, and 30 mM MgCl2, or for the ligand-free enzyme crystals from 1.2 M (NH4)2SO4, 100 mM Bis-Tris buffer pH 5.5, with 1-2% MPD, with 2-3 mg/ml protein, X-ray diffraction structure determination and analysis at 2.3 A and 1.9 A resolution, respectively
in complex with 5-fluorouridine, to 2.3 A resolution. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. A loop toward the back end of the uracil binding pocket flexibly adjusts to the varying chemistry of different compounds through an ‘induced-fit association mechanism
two crystallographic structures of human isoform UPP2 in distinctly active and inactive conformations, to 2.0 and 1.54 A resolution, respectively. The structures reveal that a conditional intramolecular disulfide bridge can form within the protein that dislocates critical phosphate-coordinating arginine residue R100 away from the active site, disabling the enzyme. The state of the disulfide bridge has further structural consequences for one face of the enzyme that suggest UPP2 may have additional functions in sensing and initiating cellular responses to oxidative stress
purified recombinant PcUP1 ligand-free and in complex with uracil/ribose-1-phosphate, 2'-deoxyuridine/phosphate or thymidine/phosphate, sitting drop vapor diffusion method, mixing of 0.001 ml of SeMet-PcUP1 as well as native PcUP1 protein at 10 mg/ml with 0.001 ml of reservoir ssolution containing 0.1 M HEPES sodium, pH 7.5, 0.8 M NaH2PO4, and 0.8 M KH2PO4, and equilibration against 0.150 ml of reservoir solution at 10°C. Crystallization of SeMet-PcUP1 and PcUP1 is completed within 5 and 3 days respectively. The PcUP1 crystals in complex with ligands are obtained by soaking crystals with mother liquor containing 10 mM each compound for 24 h, X-ray diffraction structure determination and analysis at 1.57-2.0 A resolution, molecular replacement
modeling of the inhibitors 1-((2-pyrrolidine-1-yl)ethyl)uracil and 3-((2-pyrrolidine-1-yl)ethyl)uracil into the active site of the protein, PDB entry 1NW4, by superposition of the uracil ring and the purine ring of Immucillin-H. Both compounds can fit adequately in the active site. In both cases the uracil base is predicted to be oriented in the nucleoside binding pocket by a pi-stacking similar to the purine base of Immucillin-H. The N1 of the uracil base in 1-((2-pyrrolidine-1-yl)ethyl)uracil and the N3 of the uracil base in 3-((2-pyrrolidine-1-yl)ethyl)uracil roughly superimpose with the C4 of Immucillin-H
complexed with 2,2-anhydrouridine, phosphate and potassium ions at 1.86 A resolution. The monomer is an alpha/beta-class polypeptide with a trilayer alpha/beta/alpha sandwich architecture. The potassium ion is located in the intermonomeric region of each homodimer on the local axis of second order of point group 32. The side chains of Glu49B and Ser73B and the carbonyl O atom of Ile69B in the B subunit, as well as symmetrical residues from the D subunit of the BD homodimer, coordinate K+. The residues in the phosphate binding site are Arg30B, Arg91B, Thr94B and Gly26B from the B subunit, and Arg48D from the D subunit
enzyme in complex with 5-fluorouracil, hanging-drop vapour-diffusion method, 0.5 ml of reservoir solution containing 0.34 ml 0.1 M Tris-maleate/NaOH buffer, pH 5.5, and 0.16 ml 40% w/v PEG 3350 is mixed with 2 ml protein solution containing 11.3 mg/ml enzyme in 10 mM Tris-HCl buffer, pH 7.3, 0.002 ml H2O, 0.0013 ml reservoir solution, 0.002 ml 100 mM 5-fluorouracil, and 0.0003 ml 2-propanol, 21°C, 1-2 weeks, X-ray diffraction structure determination and analysis at 2.2 A resolution, modeling
-
hanging drop vapour-diffusion method, 2.5 A resolution
-
hanging-drop vapour-diffusion method with PEG as precipitant, 2.9 A resolution
-
in complex with 5-fluorouridine, to 2.2 A resolution. The quaternary structure is represented by a hexamer comprised of six identical subunits. The packing of subunits in the hexamer can be described by the symmetry point group L33L2. The main structural and functional unit in the ligand-free state is a homodimer.The hexameric structure is formed by three homodimers with hydrophobic and hydrogen-bond interactions
in complex with with the inhibitor 2,2'-anhydrouridine, the substrate phosphate, and with both the inhibitor 2,2'-anhydrouridine and the substrate phosphate, to 2.38, 1.5 and 1.75 A resolution, respectively. The presence of the phosphate ion in the phosphate-binding site substantially changes the orientations of the side chains of the amino-acid residues Arg30, Arg91, and Arg48 coordinated to this ion. The highly flexible loop L9 is unstable
free isoform UPa and in complex with thymidine, uracil, thymine or 5-fluorouracil, sitting drop vapor diffusion method, using 100 mM trisodium citrate pH5.6, 16% (w/v) PEG4000 and 17.5% (v/v) isopropanol or 100 mM bis-Tris pH 5.5, 25% (w/v) PEG3350 and 200 mM ammonium sulfate
free enzyme and in complex with uridine, hanging drop vapor diffusion method, using 0.75 M ammonium sulfate, 0.075 M bis-Tris pH 5.5, 0.75% (w/v) PEG 3350, and 25% (v/v) glycerol
in complex with uridine, hanging drop vapor diffusion method, using
mutant enzyme C212S, hanging drop vapor diffusion method, using 0.2 M ammonium sulfate, 0.1 M Tris pH 8.5, and 25% (w/v) PEG 3350
structure analysis of the active center of enzyme UDP from Shewanella oneidensis strain MR-1 in complex with uridine and sulfate (PDB ID 4R2W)
to a resolution of 1.9 A from crystals of the free form grown on earth, 1.6 A from crystals of the complex with uridine and 0.95 A from crystals of the free form grown under microgravity. All crystals belong to the space group P21 and have similar unit-cell parameters
free selenomethionine-labeled enzyme, or enzyme in complex with uridine, X-ray diffraction structure determination and analysis at 2.7 A and 1.44 A resolution, respectively
in complex with 6-methyluracil, hanging drop vapor diffusion method, using 15% (w/v) PEG 4000, 0.1 M MgCl2, 0.1 M Tris-HCl pH 8.5
in complex with thymidine, crystals diffrat to 2.1 A resolution, space group P21
purified enzyme in complex with 2,2-anhydrouridine, X-ray diffraction structure determination and analysis at 1.34 A resolution, molecular replacement method using the structure of VchUPh in complex with 6-methyluracil (PDB ID 4K6O) as the starting model, from which all ligands, including enzyme-bound water molecules, are removed. The inhibitor molecules are located in all six active sites of the hexameric UPh molecule
-
purified native enzyme uridine phosphorylase from Vibrio cholerae in complexes with uridine, thymidine, uracil, thymine, and phosphate anion, hanging drop vapor diffusion technique, mixing of 0.002 ml of 15 mg/ml protein in 20 mM Tris-HCl, pH 7.5, 20 mM NaCl, and ligand, with 0.002 ml of reservoir solution containing 0.2 M MgCl2 hexahydrate, 15% w/v PEG 4000, 0.1 M Tris-HCl, pH 8.5, and with 0.001 ml of 0.1 M thymidine solution, for the enzyme-phosphate complex, sitting drop vapour diffusion method is used mixing 0.001 ml of the same protein solution with 0.001 ml of reservoir solution containing 0.5 M sodium diphosphate, 0.1 M ammonium sulfate, and 0.5 M potassium diphosphate, pH 7.5, 1 week, 18°C, X-ray diffraction structure determiantion and analysis at 1.29-2.24 A resolution, modeling
-
purified recombinant enzyme in complex with 6-methyluracil, mixing of 0.0015 ml of 15 mg/ml protein in Tris-HCl buffer with 0.0015 ml of reservoir solution containing 0.2 M ?gCl2 hexahydrate, 15% w/v PEG 4000, and 0.1 M Tris-HCl, pH 8.5, as well as with 0.001 ml of 6-methyluracil solution, 1 week, X-ray diffraction structure determination and analysis at 1.17 A resolution
-
in silico model of the complex with 5-fluorouracil. The quaternary structure is represented by a hexamer comprised of six identical subunits. The packing of subunits in the hexamer can be described by the symmetry point group L33L2. The hexameric structure of the complex is stabilized by hydrophobic interactions and hydrogen bonds between the amino-acid residues of adjacent homodimers
-
sitting drop vapor diffusion method, using 1 M Tris, 5% (w/v) polyglutamic acid, 20% (w/v) PEG 2000, pH 7.8