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

Abstract

Discovery R&D, Dow AgroSciences, Indianapolis, Indiana 46268 Inositol 1,3,4,5,6-pentakisphosphate 2-kinase, an enzyme encoded by the gene IPK1, catalyzes the terminal step in the phytic acid biosynthetic pathway. We report here the isolation and characterization of IPK1 cDNA and genomic clones from maize (Zea mays). DNA Southern-blot analysis revealed that ZmIPK1 in the maize genome constitutes a small gene family with two members. Two nearly identical ZmIPK1 paralogs, designated as ZmIPK1A and ZmIPK1B, were identified. The transcripts of ZmIPK1A were detected in various maize tissues, including leaves, silks, immature ears, seeds at 12 d after pollination, midstage endosperm, and maturing embryos. However, the transcripts of ZmIPK1B were exclusively detected in roots. A variety of alternative splicing products of ZmIPK1A were discovered in maize leaves and seeds. These products are derived from alternative acceptor sites, alternative donor sites, and retained introns in the transcripts. Consequently, up to 50% of the ZmIPK1A transcripts in maize seeds and leaves have an interrupted open reading frame. In contrast, only one type of splicing product of ZmIPK1B was detected in roots. When expressed in Escherichia coli and subsequently purified, the ZmIPK1 enzyme catalyzes the conversion of myo-inositol 1,3,4,5,6-pentakisphosphate to phytic acid. In addition, it is also capable of catalyzing the phosphorylation of myo-inositol 1,4,6-trisphosphate, myo-inositol 1,4,5,6-tetrakisphosphate, and myo-inositol 3,4,5,6-tetrakisphosphate. Nuclear magnetic resonance spectroscopy analysis indicates that the phosphorylation product of myoinositol 1,4,6-trisphosphate is inositol 1,2,4,6-tetrakisphosphate. Kinetic studies showed that the K m for ZmIPK1 using myo-inositol 1,3,4,5,6-pentakisphosphate as a substrate is 119 mM with a V max at 625 nmol/min/mg. These data describing the tissue-specific accumulation and alternative splicing of the transcripts from two nearly identical ZmIPK1 paralogs suggest that maize has a highly sophisticated regulatory mechanism controlling phytic acid biosynthesis.

Conformational Changes in Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase upon Substrate Binding

“…This work is focused on the study of one of those kinases, the inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP 5 2-K, IPK1), an enzyme that catalyzes the synthesis of a crucial compound, inositol 1,2,3,4,5,6-hexakisphosphate (IP 6 ), by phosphorylation of the axial 2-OH of IP 5 . IP 5 2-K has been cloned and characterized from many organisms, from yeast to humans (3)(4)(5)(6)(7)(8).…”

“…Ipk1 gene disruption in mice results in early lethality of the embryos (9). IP 6 participates in mRNA export (10), regulation of chromatin state (11), developmental processes (12), and apo-ptosis (13). In addition, IP 6 serves as substrate for the synthesis of diphosphoinositol polyphosphates (inositol pyrophosphates), emergent molecules with multiple functions (14).…”

“…IP 5 2-K has been cloned and characterized from many organisms, from yeast to humans (3)(4)(5)(6)(7)(8). Ipk1 gene disruption in mice results in early lethality of the embryos (9).…”

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase is a distant IPK member with a singular inositide binding site for axial 2-OH recognition

“…The N-lobe helps to inositide binding by coordinating P1 and P3. The strong interaction that all IP 5 phosphates establish with the protein explains why other less substituted InsPs lacking phosphates on 1-, 3-, or 5-or 3,5-positions are substrates of IP 5 2-K (20,24,31).…”

“…The family of enzymes that catalyzes the phosphorylation of IP 5 to form IP 6 are known as Ipk1, after the ortholog first identified in yeast (3) or InsP 5 2-kinases (IP 5 2-Ks), after the enzyme activity characterized from various sources (19)(20)(21)(22)(23)(24). It is only recently, however, that important physiological roles other than a biosynthetic enzyme have been ascribed to IP 5 2-K.…”