Sun Yuejin, Thompson Mark, Lin Gaofeng, Butler Holly, Gao Zhifang, Thornburgh Scott, Yau Kerrm, Smith Doug A, Shukla Vipula K
Discovery R&D, Dow AgroSciences, Indianapolis, IN 46268, USA.
Plant Physiol. 2007 Jul;144(3):1278-91. doi: 10.1104/pp.107.095455. Epub 2007 May 25.
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.
肌醇1,3,4,5,6 - 五磷酸2 - 激酶是一种由IPK1基因编码的酶，催化植酸生物合成途径的最后一步。我们在此报告从玉米（Zea mays）中分离和鉴定IPK1 cDNA及基因组克隆。DNA Southern杂交分析表明，玉米基因组中的ZmIPK1构成一个有两个成员的小基因家族。鉴定出两个几乎相同的ZmIPK1旁系同源基因，命名为ZmIPK1A和ZmIPK1B。在玉米的各种组织中检测到ZmIPK1A的转录本，包括叶片、花丝、未成熟的果穗、授粉后12天的种子、中期胚乳和成熟胚。然而，仅在根中检测到ZmIPK1B的转录本。在玉米叶片和种子中发现了多种ZmIPK1A的可变剪接产物。这些产物源自转录本中的可变受体位点、可变供体位点和保留的内含子。因此，玉米种子和叶片中高达50%的ZmIPK1A转录本具有中断的开放阅读框。相比之下，在根中仅检测到一种类型的ZmIPK1B剪接产物。当在大肠杆菌中表达并随后纯化时，ZmIPK1酶催化肌醇1,3,4,5,6 - 五磷酸转化为植酸。此外，它还能够催化肌醇1,4,6 - 三磷酸、肌醇1,4,5,6 - 四磷酸和肌醇3,4,5,6 - 四磷酸的磷酸化。核磁共振光谱分析表明，肌醇1,4,6 - 三磷酸的磷酸化产物是肌醇1,2,4,6 - 四磷酸。动力学研究表明，以肌醇1,3,4,5,6 - 五磷酸为底物时，ZmIPK1的K(m)为119微摩尔，V(max)为625纳摩尔/分钟/毫克。这些描述两个几乎相同的ZmIPK1旁系同源基因转录本的组织特异性积累和可变剪接的数据表明，玉米具有高度复杂的调控机制来控制植酸的生物合成。
