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

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‐pentakisphosphate (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 IP 5 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 P 2 1 2 1 2, 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.