A Fluorescent Probe Identifies Active Site Ligands of Inositol Pentakisphosphate 2‑Kinase

Inositol
pentakisphosphate 2-kinase catalyzes the phosphorylation
of the axial 2-OH of myo-inositol 1,3,4,5,6-pentakisphosphate
for de novo synthesis of myo-inositol hexakisphosphate.
Disruption of inositol pentakisphosphate 2-kinase profoundly influences
cellular processes, from nuclear mRNA export and phosphate homeostasis
in yeast and plants to establishment of left–right asymmetry
in zebrafish. We elaborate an active site fluorescent probe that allows
high throughput screening of Arabidopsis inositol
pentakisphosphate 2-kinase. We show that the probe has a binding constant
comparable to the Km values of inositol
phosphate substrates of this enzyme and can be used to prospect for
novel substrates and inhibitors of inositol phosphate kinases. We
identify several micromolar Ki inhibitors
and validate this approach by solving the crystal structure of protein
in complex with purpurogallin. We additionally solve structures of
protein in complexes with epimeric higher inositol phosphates. This
probe may find utility in characterization of a wide family of inositol
phosphate kinases.