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Results 1 - 8 of 8
EC Number
Crystallization
Reference
enzyme in complex with adenosine-5'-monophosphate and a phosphate ion, crystallization at 4°C by hanging-drop vapor-diffusion method
enzyme, 10 mg/ml, in complex with 9-deazaadenine and sulfate or Mg-phosphoribosyldiphosphate, 50 mM Hepes, pH 6.0, 8 mM MgCl2, 1 mM DTT, 1:2 molar ratio of 9-deazaadenine and iminoribitol, 1 mM sodium diphosphate, after 45 min incubation preparation of crystallization drops, crystals are obtained from mother liquid 0.1 M sodium acetate, pH 4.6, 24% polyethylene glycol 4000, 0.2 M ammonium sulfate, 0.05 M urea, 18°C, X-ray diffraction structure analysis, hydrogen bond network in the complexes
four crystal structures: (1) a structure (the enzyme/Pi complex) refined at 2.4 A with inorganic phosphate or sulfate bound in the 5-phosphoribosyl binding pocket, (2) an adenine bound structure (the enzyme/adenine complex) refined at 2.4 A, which shows adenine together with phosphates both at the 5'-phosphoryl and PPi positions of the presumed PRPP binding site, (3) an AMP bound structure (the enzyme/AMP complex) refined at 2.4 A, and (4) an ADP bound structure (the enzyme/ADP complex), refined at 2.8 A containing the inhibitor ADP bound like AMP with both the alpha- and beta-phosphates occupying the 5'-phosphoribosyl binding site. No crystals of the enzyme in complex with 5-phosphoribosyl-alpha-1-pyrophosphate are obtained, likely because the enzyme catalyzes a slow breakdown of 5-phosphoribosyl-alpha-1-pyrophosphate to ribose 5-phosphate and PPi. The crystal structure suggests that the enzyme evolves from a 6-oxopurine phosphoribosyltransferase. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known adenine phosphoribosyltransferases implying that adenine phosphoribosyltransferase functionality in Crenarchaeotae has its evolutionary origin in this family of 6-oxopurine phosphoribosyltransferases. The N-terminal two-thirds of the polypeptide chain folds as a traditional type I PRTase with a five-stranded beta-sheet surrounded by helices. The C-terminal third adopts an unusual three-helix bundle structure that together with the nucleobase-binding loop undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site
hanging drop vapour diffusion method, with 15% (v/v) glycerol, 25.5% (w/v) PEG 4000, 0.17 M sodium acetate, and 0.085 M Tris-HCl, pH 8.5
mixing of protein solution 13-15 mg/ml with an equal volume of mother liquid 0.1 M Hepes, pH 7.5, 1.5 M lithium sulfate, then equilibration against mother liquid at 18°C, crystals appear after 3 days, X-ray diffraction structure analysis, also crystallization of the enzyme in presence of diphosphate, Mg2+ or inhibitor immucillin, which do not bind at the active site
purified recombinant native and selenomethionine-labeled enzymes, sitting drop vapour diffusion method, 0.001 ml of 20 mg/ml native protein in 20 mM Tris-HCl, 50 mM NaCl, pH 8.0, is mixed with an equal volume of reservoir solution containing 100 mM MES, pH 5.5, 100 mM calcium acetate, 3% w/v PEG 10000, and 3% v/v MeOH, 1 week, for the selenomethionine-labeled protein 40 mM calcium acetate, 1.5% w/v PEG 10000, and 3% v/v MeOH is used at a protein concentration of 29.4 mg/ml, X-ray diffraction structure determination and analysis at 1.9-2.6 A resolution, multiple wavelength anomalous dispersion method, asymmetric unit of two pairs of identical dimers, each related by noncrystallographic two-fold symmetry, a fifth monomer forms a similar dimer across a crystallographic two-fold axis, modeling
recombinant human APRT is crystallized in complex with adenosine 5'-monophosphate, hanging-drop vapour-diffusion method
vapour diffusion method, hanging drops from solution: 10 mg/ml purified apo-enzyme in 10 mM MES, pH 6.0, 1 mM dithiothreitol, 5 mM MgCl2, 4°C, reservoir solution: 7-11% polyethylene glycol 5000 monomethyl ether, 0.2 M ammonium acetate, 0.1 M sodium citrate, pH 4.9, 10 mM MgCl2, 1.2-1.6 M ammonium sulfate, for AMP- or adenine-bound crystals addition of 10 mM AMP or 5 mM adenine in the reservoir solution, structure analysis
Results 1 - 8 of 8