A minimum catalytic unit for synthesis of InsP6 and 5-PP-InsP5 in <i>Arabidopsis</i>

Inositol pyrophosphates (diphosphoinositol phosphates) are reported agents of phosphate homeostasis, disease resistance, and hormone action in plants. Of the enzymes that have been shown to synthesize inositol pyrophosphates, inositol tris/tetrakisphosphate (ITPK)1 and Arabidopsis thaliana diphosphoinositol pentakisphosphate kinase (VIH)1/VIH2 share the ATP-grasp fold-the latter also possesses a phosphatase domain. Among ATP-grasp inositol phosphate kinases, ITPK1 is particularly flexible-phosphorylating equatorial hydroxyls and equatorial phosphates on inositol phosphates. Herein, we show that the combination of ITPK1 and inositol pentakisphosphate 2-kinase (IPK1) is sufficient to synthesize 5-PP-InsP5 from 1D-myo-inositol 3-monophosphate (Ins3P) and that ITPK1 is capable of converting 1D-myo-inositol 1-monophosphate to myo-inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,5,6)P5]. In defining a minimal catalytic unit for synthesis of both myo-inositol 1,2,3,4,5,6-hexakisphosphate (InsP6/Ins(1,2,3,4,5,6)P6) and 5-PP-InsP5, we define the minimum enzymology of the 'lipid-independent' pathway of InsP6 synthesis from Ins3P and its intermediates. The pathway proceeds: Ins3P, 1D-myo-inositol 3,4-bisphosphate, 1D-myo-inositol 3,4,5-trisphosphate, 1D-myo-inositol 3,4,5,6-tetrakisphosphate, Ins(1,3,4,5,6)P5, Ins(1,2,3,4,5,6)P6, and therefrom to 5-diphosphoinositol-1,2,3,4,6-pentakisphosphate [5-PP-Ins(1,2,3,4,6)P5].