Structural and biochemical studies of plant inositol phosphate kinases
Among inositol phosphates, inositol hexakisphosphate constitutes the main form of phosphorus storage in seeds and beans. However, the pathway(s) of inositol hexakisphosphatesynthesis, particularly in plants, remain incomplete. To further our understanding of these pathways, five inositol phosphate kinases: AtIPK1, AtITPK2, AtITPK4, StIPMK and StIPKα, along with two inositol Phosphate binding proteins: COI1 and TIR1, were cloned with the aim of fully characterising each. The research became centered around AtIPK1, although AtITPK4 was also investigated as a novel ITPK family member. X-ray crystallographic structures of AtIPK1XHis in complex with myo-inositol hexakisphosphate, myo-inositol 1,3,4,5,6-pentakisphosphate, neo- and D-chiro-inositol pentakisphosphate were collected to aid understanding of substrate use. Structural data showed that AtIPK1 was able to form a complex with these substrates. Enzyme assays revealed that neo-inositol 1,3,4,6-tetrakisphosphate and D-chiro-inositol 2,3,4,5-tetrakisphosphate are phosphorylated twice by AtIPK1, producing neo-inositol hexakisphosphate and D-chiro-inositol hexakisphosphate. The enzyme was shown to catalyze phosphotransfer reactions in the reverse direction. The reactions shown to be catalyzed by AtIPK1 afford potential explanation of the range of inositol phosphate Stereoisomers found in soils. Solved crystal structures of binary and ternary complexes of AtIPK1, nucleotide and individual benzene tetrakisphosphates, a group of novel compounds which have been proposed as structural mimics of inositol phosphates, revealed that AtIPK1 binds these inositol phosphate mimics. Complementary enzyme assays showed no evidence of addition or removal of phosphates from these mimics. Further assays demonstrated that benzene phosphates are inhibitors of AtIPK1, offering potential for the design of stronger inhibitors of this enzyme. AtIPK1 contains a zinc ion coordinated by a zinc finger motif that is conserved in all plant IPK1 homologs, but is not homologous to that found in any plant enzyme, or indeed in any other organism. On oxidative stress treatment in vitro, AtIPK1 appeared to release the zinc and became unstable. Confocal microscopy studies of IPK1:GFP constructs in Nicotiana benthamiana suggested that oxidative stress, or mutation of the zinc coordinating residues, alters AtIPK1 localisation and results in apparent aggregation of the protein. DNA binding studies, using agarose gel electrophoresis, and fluorescence polarisation studies, using fluorescent nucleotide probes, suggested that AtIPK1 binds DNA in a non sequence-specific manner. Identification of the nucleotide target(s) of AtIPK1, as well as the effect of oxidative stress on DNA binding, are likely to be key to determining whether this intriguing enzyme plays a role in cell signalling beyond simply regulating levels of IP6. At ITPK4, a member of the inositol tetrakisphosphate family, contained a novel additional N-terminal sequence not present in other members of this family as well as a distinctly different kinase activity to other family members. It was therefore cloned for structural studies along with N- and C-terminal regions of the protein to attempt to understand the function of the protein.
Networked Digital Library of Theses and Dissertations