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

The metabolic pathway(s) by which plants synthesize InsP(6) (inositol 1,2,3,4,5,6-hexakisphosphate) remains largely undefined [Shears (1998) Biochim. Biophys. Acta 1436, 49-67], while the identifies 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, Medley and Ert1 (2003) Plant Physiol. 131, 507-515], unidentified. A yeast enzyme, SclPK1, catalyses the synthesis of InsP(6) by 2-phosphorylation of Ins( 1,3,4,5,6)P-5 (inositol 1,3,4,5,6-pentakisphosphate). A human orthologue, Hs1PK1, is able to substitute for yeast Sc1PK1, restoring InsP(6) production Saccharomyces cerevisiae Mutant strain lacking the Sc1PK1 open reading frame (Sc1pk1 Delta). We have identified an Arabidopsis genomic sequence, At1PK1, encoding an Ins(1,3,4,5,6)P-5 2-kinase. Inclusion of the At1PK1 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 ail additional motif. Recombinant At1PK1 protein expressed ill Escherichia coli catalysed the synthesis of InsP, from Ins(1,3,4,5,6)P-5. The enzyme obeyed Michaelis-Menten kinetics with ail apparent V-max of 35 nmol (.) min(-1 .) (mg of protein)(-1) and a K for Ins(1,3,4,5,6)P-5 of 22 mu M at 0.4 mM ATP. RT (reverse transcriptase)-PCR analysis of At1PK1 transcripts revealed that At1PK1 is expressed in siliques, leaves and cauline leaves. In situ hybridization experiments further revealed strong expression of At1PK1 in male and female organs of flower birds. Expression of At1PK1 protein in an Sc1pk1 Delta mutant Strain restored InsP(6) production and rescued the temperature-sensitive growth phenotype of the yeast.