Expression, purification, crystallization and preliminary X-ray diffraction analysis of the apo form of InsP 5 2-K from Arabidopsis thaliana

Jose Ignacio Bãos-Sanz, Julia Sanz-Aparicio, Charles A. Brearley, Beatriz González

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2 Citations (Scopus)

Abstract

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP 5 2-K) is a key enzyme that catalyzes the synthesis of phytic acid (IP 6) from inositol 1,3,4,5,6-pentakisphos-phate (IP 5) and ATP. The first structure of IP 5 2-K, that from Arabidopsis thaliana, has been solved previously; it only crystallized in the presence of inositol, either the substrate IP 5 or the product IP 6, and failed to crystallize in its free state (without inositol). Based on structural analysis, a point mutation of IP5 2-K (W129A) has been produced in order to overcome this limitation and obtain information about protein conformational changes upon substrate binding. Here, the production and crystallization of W129A IP 5 2-K in its free state and with bound nucleotide is described. These crystals differed from the native crystals and belonged to the orthorhombic space group P2 12 12, with unit-cell parameters a = 66.00, b = 68.23, c = 105.80 Å and a = 63.06, b = 71.80, c = 100.23 Å, respectively. The crystals diffracted to resolutions of 2.22 Å (apo) and 2.05 Å (nucleotide bound) using synchrotron radiation and contained one molecule per asymmetric unit. The structures have been determined using the molecular-replacement method and refinement is being undertaken.

Original languageEnglish
Pages (from-to)701-704
Number of pages4
JournalActa Crystallographica Section F: Structural Biology and Crystallization Communications
Volume68
Issue number6
DOIs
Publication statusPublished - 1 May 2012

Keywords

  • inositol kinases
  • inositol phosphate
  • IP 2-K
  • IP
  • phytic acid

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