Substrate Promiscuity of Inositol 1,4,5-trisphosphate Kinase Driven by Structurally-modified Ligands and Active Site Plasticity

D-myo-inositol 1,4,5-trisphosphate (InsP) is a fundamental second messenger in cellular Ca mobilization. InsP 3-kinase, a highly specific enzyme binding InsP in just one mode, phosphorylates InsP specifically at its secondary 3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biology approach with both synthetised and established ligands, combining synthesis, crystallography, computational docking, HPLC and fluorescence polarization binding assays using fluorescently-tagged InsP, we have surveyed the limits of InsP 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticity generating a helix-tilt. These facilitated uncovering of unexpected substrates phosphorylated at a surrogate extended primary hydroxyl at the inositol pseudo 3-position, applicable even to carbohydrate-based substrates. Crystallization experiments designed to allow reactions to proceed in situ facilitated unequivocal characterization of the atypical tetrakisphosphate products. In summary, we define features of InsP 3-kinase plasticity and substrate tolerance that may be more widely exploitable.