Inositol 1,3,4,5,6-pentakisphosphate 2-kinase from maize: molecular and biochemical characterization.
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 myo-inositol 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 microm 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.
Sun
Yuejin
Y
Discovery R&D, Dow AgroSciences, Indianapolis, IN 46268, USA. ysun@dow.com
Thompson
Mark
M
Lin
Gaofeng
G
Butler
Holly
H
Gao
Zhifang
Z
Thornburgh
Scott
S
Yau
Kerrm
K
Smith
Doug A
DA
Shukla
Vipula K
VK
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D010833:Phytic Acid / Q000096:biosynthesis*; D018515:Plant Leaves / Q000201:enzymology*; D018517:Plant Roots / Q000201:enzymology; D012639:Seeds / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D010833:Phytic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); C502806:inositol 1,3,4,5,6-pentakisphosphate 2-kinase