Crystallization and preliminary X-ray diffraction analysis of recombinant phosphoribosylpyrophosphate synthetase from th
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CTURE OF MACROMOLECULAR COMPOUNDS
Crystallization and Preliminary X-Ray Diffraction Analysis of Recombinant Phosphoribosylpyrophosphate Synthetase from the Thermophilic Thermus Thermophilus Strain HB27 Yu. A. Abramchika, V. I. Timofeevb,c,*, T. I. Muravievaa, E. V. Sinitsynaa, R. S. Esipova,**, and I. P. Kuranovab,c,*** a
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia b Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics”, Russian Academy of Sciences, Moscow, 119333 Russia c National Research Centre “Kurchatov Institute,” Moscow, 123098 Russia *e-mail: [email protected] **e-mail: [email protected] ***e-mail: [email protected] Received May 26, 2016
Abstract—Phosphoribosylpyrophosphate synthetases (PRPP synthetases) are among the key enzymes essential for vital functions of organisms and are involved in the biosynthesis of purine and pyrimidine nucleotides, coenzymes, and the amino acids histidine and tryptophan. These enzymes are used in biotechnology for the combined chemoenzymatic synthesis of natural nucleotide analogs. Recombinant phosphoribosylpyrophosphate synthetase I from the thermophilic strain HB27 of the bacterium Thermus thermophilus (T. th HB27) has high thermal stability and shows maximum activity at 75°С, due to which this enzyme holds promise for biotechnological applications. In order to grow crystals and study them by X-ray crystallography, an enzyme sample, which was produced using a highly efficient producer strain, was purified by affinity and gel-filtration chromatography. The screening of crystallization conditions was performed by the vapor-diffusion technique. The crystals of the enzyme suitable for X-ray diffraction were grown by the counter-diffusion method through a gel layer. These crystals were used to collect the X-ray diffraction data set at the SPring-8 synchrotron radiation facility (Japan) to 3-Å resolution. The crystals belong to sp. gr. P21 and have the following unitcell parameters: a = 107.7 Å, b = 112.6 Å, c = 110.2 Å, α = γ = 90°, β = 116.6°. The X-ray diffraction data set is suitable for determining the three-dimensional structure of the enzyme at 3.0-Å resolution. DOI: 10.1134/S1063774517010035
INTRODUCTION Enzymes belonging to the posphoribosylpyrophosphate synthetase family (PRPP synthetases, EC 2.7.6.1) catalyze the synthesis of 5-phosphoribosyl 1-pyrophosphate (5-PRPP) from adenosine triphosphate and ribose 5-phosphate [1]. The product of the catalyzed reaction (5-PRPP) is an intermediate in a number of important metabolic pathways of synthesis of purine and pyrimidine nucleotides, coenzymes, and the amino acids histidine and tryptophan and is among metabolites permanently required by the cell [2–6]. In some bacteria, more than a dozen enzymes utilize 5-PRPP as the substrate. Therefore, PRPP synthetases are among the key enzymes ensuring the viability of the organism [7]. Enzymes involved in nucleotide metabolism are of considerable interest for biotech
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