Characterization of an Arabidopsis inositol 1,3,4,5,6-pentakisphosphate 2-kinase (AtIPK1)

Abstract

The metabolic pathway(s) by which plants synthesize InsP6 (inositol 1,2,3,4,5,6-hexakisphosphate) remains largely undefined [Shears (1998) Biochim. Biophys. Acta 1436, 49-67], while the identities of the genes that encode enzymes catalysing individual steps in these pathways are, with the notable exception of myo-inositol phosphate synthase and ZmIpk [Shi, Wang, Wu, Hazebroek, Meeley and Ertl (2003) Plant Physiol. 131, 507-515], unidentified. A yeast enzyme, ScIPK1, catalyses the synthesis of InsP6 by 2-phosphorylation of Ins(1,3,4,5,6)P5 (inositol 1,3,4,5,6-pentakisphosphate). A human orthologue, HsIPK1, is able to substitute for yeast ScIPK1, restoring InsP6 production in a Saccharomyces cerevisiae mutant strain lacking the ScIPK1 open reading frame (ScIpk1Delta). We have identified an Arabidopsis genomic sequence, AtIPK1, encoding an Ins(1,3,4,5,6)P5 2-kinase. Inclusion of the AtIPK1 protein in alignments of amino acid sequences reveals that human and Arabidopis kinases are more similar to each other than to the S. cerevisiae enzyme, and further identifies an additional motif. Recombinant AtIPK1 protein expressed in Escherichia coli catalysed the synthesis of InsP6 from Ins(1,3,4,5,6)P5. The enzyme obeyed Michaelis-Menten kinetics with an apparent V(max) of 35 nmol x min(-1) x (mg of protein)(-1) and a K(m) for Ins(1,3,4,5,6)P5 of 22 microM at 0.4 mM ATP. RT (reverse transcriptase)-PCR analysis of AtIPK1 transcripts revealed that AtIPK1 is expressed in siliques, leaves and cauline leaves. In situ hybridization experiments further revealed strong expression of AtIPK1 in male and female organs of flower buds. Expression of AtIPK1 protein in an ScIpk1Delta mutant strain restored InsP6 production and rescued the temperature-sensitive growth phenotype of the yeast.

Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase from Maize: Molecular and Biochemical Characterization

“…The apparent K m for Ins(1,3,4,5,6)P 5 and V max were derived from the plot as 119 mM and 625 nmol/min/mg, respectively. Both K m and V max of ZmIPK1 are significantly higher than that reported for the enzyme from Arabidopsis (Stevenson-Paulik et al, 2005;Sweetman et al, 2006). This difference may reflect the catalytic characteristics of the IPK1 kinase from the two divergent plant species.…”

“…Figure 1 shows the alignment of the predicted amino acid sequences of IPK1 gene products from maize, Arabidopsis, apple, Sorghum, and rice. The conserved motifs known as A, B, C, D, and E boxes, identified in the IPK1 proteins from human (Verbsky et al, 2002) and Arabidopsis (Sweetman et al, 2006), are present in the monocot species examined. In addition, domains we designate as F and G boxes were conserved in such diverse higher plants as Arabidopsis and apple (dicots) and rice, Sorghum and maize (monocots).…”

“…Identical amino acid residues are marked with black background and the conserved residues with gray background. The conserved boxes A, B, C, and D, reported in Arabidopsis and human IPK1 proteins (Verbsky et al, 2002;Sweetman et al, 2006), and the conserved E and F domains, are labeled above the relevant sequences. (Fig.…”

“…The Michaelis-Menten kinetics of ZmIPK1 protein was investigated according to experimental conditions described by Sweetman et al (2006). To determine the K m for Ins(1,3,4,5,6)P 5 , 400 mM of ATP was used in the reactions.…”

“…More recently, progress on characterization of this kinase at the molecular level has followed the discovery of the Ins(1,3,4,5,6)P 5 2-kinase gene (IPK1) in budding yeast (York et al, 1999;Ives et al, 2000). Orthologs of IPK1 were subsequently isolated from Schizosaccharomyces pombe (Ives et al, 2000), human (Verbsky et al, 2002), Drosophila (Seeds et al, 2004), and Arabidopsis (Stevenson-Paulik et al, 2005;Sweetman et al, 2006). T-DNA insertional disruption of the Arabidopsis AtIPK1 gene was shown to result in accumulation of Ins (1,3,4,5,6)P 5 in seeds, and Arabidopsis lines containing insertional disruptions in both AtIPK1 and AtIPK2b gave rise to phytate-free seeds (Stevenson-Paulik et al, 2005).…”