Expression, purification, crystallization and preliminary X-ray diffraction analysis of the apo form of InsP5 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 P2(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.
Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain.
Jose Ignacio
JI
Baños-Sanz
Julia
J
Sanz-Aparicio
Charles A
CA
Brearley
Beatriz
B
González
D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017360:Arabidopsis / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D001051:Apoenzymes; D017853:Phosphotransferases (Alcohol Group Acceptor); C502806:inositol 1,3,4,5,6-pentakisphosphate 2-kinase