title: 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.
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
authors.forename: Dylan
authors.initials: D
authors.lastname: Sweetman
authors.forename: Sue
authors.initials: S
authors.lastname: Johnson
authors.forename: Samuel E K
authors.initials: SE
authors.lastname: Caddick
authors.forename: David E
authors.initials: DE
authors.lastname: Hanke
authors.forename: Charles A
authors.initials: CA
authors.lastname: Brearley
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D035264:Flowers / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D029681:Arabidopsis Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C508906:inositol 1,3,4,5,6-pentakisphosphate 2-kinase, Arabidopsis