Characterization of a cDNA encoding Arabidopsis thaliana inositol 1,3,4-trisphosphate 5/6-kinase.

We have sequenced and recombinantly expressed as a fusion protein an expressed sequence tag clone (GB Z25963) from Arabidopsis thaliana that represents the plant homologue of human inositol 1,3,4 trisphosphate 5/6-kinase. The 1365 base pair clone has an open reading frame of 960 base pairs that predicts a protein product of 36.2 kDa, with a pI of 6.1. There is no polyadenylation signal or poly (A) tail, suggesting that additional 3' sequence remains to be identified. The amino acid sequence is 30% identical to the human protein. There are several short regions with particularly high degrees of identity between the human and Arabidopsis protein sequences, and these may be useful in identifying the active site of the enzyme. The expressed sequence tag was expressed as a fusion protein in Escherichia coli, with a carboxyl terminal deletion removing one region of high identity between the two proteins. The protein product of this construct was found to have inositol 1,3,4-trisphosphate 5/6-kinase activity. The Arabidopsis enzyme produced both inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate as products in a ratio of 1:3, in contrast with the human enzyme which gives a product ratio of 3:1.