Abstract
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].