Journal article
Characterization of a multifunctional inositol phosphate kinase from rice and barley belonging to the ATP-grasp superfamily
OsIpk and HvIk, inositol phosphate kinases, were cloned from rice (Oryza sativa L. var. indica, IR64) and barley (Hordeum vulgare) respectively.. IP Sequence alignment showed that they belong to the ATP-grasp family, which includes inositol 1,3,4-trisphosphate 5/6-kinase from humans and Arabidopsis.
Residues that are binding sites for ATP and coordinate magnesium in absence or presence of inositol phosphate are conserved and in total 23 residues are invariant among the twelve aligned inositol phosphate kinases. The genes were heterologously expressed in Escherichia coli and kinase activity assays with 17 different isomers of inositol mono-/di-/tri-/tetra-/pentaphosphate as well as phytate were performed.
The strongest activity for both kinases was observed with Ins(3,4,5,6)P-4, which candidates as the primary substrate for these kinases in plants. Several species-specific differences between the two recombinant Ipks were observed. Rice OsIpk showed detectable kinase activity towards eight different substrates, whereas barley HvIpk showed kinase activity with all the substrates including inositol mono- and bisphosphates.
HvIpk showed 3-kinase activity towards the Ins (1,4,5)P-3 substrate and it also interconverted the two substrates Ins(1,3,4,5)P-4 and Ins(1,3,4,6)P-4 by isomerase activity, which was not observed for the rice homologue. Both OsIpk and HvIpk had no detectable 2-kinase activity. Furthermore, the two Ipks showed phosphatase activity towards several inositol phosphates.
Expression analysis by RT-PCR demonstrated that the Ipk gene was equally expressed in different tissues and developmental stages. Taken together, these results show that the Ipk kinase plays a significant role in the inositol phosphate interacting network in plants. (C) 2007 Elsevier B.V. All rights reserved.
Language: | English |
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Year: | 2007 |
Pages: | 114-125 |
ISSN: | 18790038 and 03781119 |
Types: | Journal article |
DOI: | 10.1016/j.gene.2007.04.018 |
ORCIDs: | 0000-0001-6169-2504 |
Amino Acid Sequence Cloning, Molecular Former LIFE faculty Gene expression Gene expression, Phosphatase, Phosphorus, Phylogeny, Phytic acid Genes, Plant HPIC; high-performance ion chromatography Hordeum Hv; Hordeum vulgare Inositol Phosphates InsP; inositol phosphate Ipk; Inositol phosphate kinase Molecular Sequence Data Oryza PA; phytic acid, Os, Oryza sativa Phosphatase Phosphorus Phosphotransferases (Alcohol Group Acceptor) Phylogeny Phytic Acid Phytic acid RT-PCR; Reverse transcription-polymerase chain reaction Recombinant Proteins Sequence Homology, Amino Acid Species Specificity Substrate Specificity aa; amino acid(s) bp; base pairs