posted on 2023-12-26, 13:05authored byHayley
L. Whitfield, Raquel Faba Rodriguez, Megan L. Shipton, Arthur W.H. Li, Andrew M. Riley, Barry V.L. Potter, Andrew M. Hemmings, Charles A. Brearley
Inositol phosphates and their pyrophosphorylated derivatives
are
responsive to the phosphate supply and are agents of phosphate homeostasis
and other aspects of physiology. It seems likely that the enzymes
that interconvert these signals work against the prevailing milieu
of mixed populations of competing substrates and products. The synthesis
of inositol pyrophosphates is mediated in plants by two classes of
ATP-grasp fold kinase: PPIP5 kinases, known as VIH, and members of
the inositol tris/tetrakisphosphate kinase (ITPK) family, specifically
ITPK1/2. A molecular explanation of the contribution of ITPK1/2 to
inositol pyrophosphate synthesis and turnover in plants is incomplete:
the absence of nucleotide in published crystal structures limits the
explanation of phosphotransfer reactions, and little is known of the
affinity of potential substrates and competitors for ITPK1. Herein,
we describe a complex of ADP and StITPK1 at 2.26
Å resolution and use a simple fluorescence polarization approach
to compare the affinity of binding of diverse inositol phosphates,
inositol pyrophosphates, and analogues. By simple HPLC, we reveal
the novel catalytic capability of ITPK1 for different inositol pyrophosphates
and show Ins(3,4,5,6)P4 to be a potent inhibitor of the
inositol pyrophosphate-synthesizing activity of ITPK1. We further
describe the exquisite specificity of ITPK1 for the myo-isomer among naturally occurring inositol hexakisphosphates.