Crystal Structure and Product Analysis of an Archaeal myo-Inositol Kinase Reveal Substrate Recognition Mode and 3-OH Phosphorylation
Crystal Structure and Product Analysis of an Archaeal myo-Inositol Kinase Reveal Substrate Recognition Mode and 3-OH Phosphorylation
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DOI：
10.1021/acs.biochem.5b00296
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发表时间：
2015-06-09
期刊：
影响因子：
2.9
通讯作者：
Miki, Kunio
中科院分区：
文献类型：
--
作者：
Nagata, Ryuhei;Fujihashi, Masahiro;Miki, Kunio
柯达热球菌产的TK2185蛋白最近被鉴定为一种催化肌醇磷酸化的酶。到目前为止，只有两个肌醇激酶被鉴定，它们是来自玉米的TK2285蛋白和Lpa3，这两个蛋白都属于核糖激酶家族。在任何一种情况下，肌醇的六个羟基中的哪一个是磷酸化的仍然是未知的。此外，人们对这些酶的肌醇结合机制知之甚少。在这项工作中，我们确定了两种晶体结构：TK2285蛋白与底物(ATP类似物和肌醇)络合的晶体结构或酶反应生成的产物的晶体结构。对三元底物复合体结构和定点突变的分析表明，有5个残基参与了与肌醇的相互作用。与其他核糖激酶家族酶的结构比较表明，这五个残基中的两个残基Q136和R140是肌醇激酶特有的。TK2285蛋白与肌醇和三磷酸腺苷作用形成的三元产物复合体的晶体结构中，活性部位含有一维肌醇3-磷酸(Ins(3)P)。用手性柱进行的核磁共振和高效液相分析也表明TK2285反应产物为Ins(3)P。结果表明，TK2285蛋白特异性催化肌醇3-羟基的磷酸化。因此，我们将TK2285命名为肌醇3-激酶(MI3K)。对反应产物的准确鉴定将为进一步研究古生代的肌醇代谢提供良好的基础。
The TK2185 protein from Thermococcus kodakarensis was recently characterized, as an enzyme catalyzing the phosphorylation of myo-inositol. Only two myo-inositol kinases have been identified so far, the TK2285 protein and Lpa3 from Zea mays, both of which belong to the ribokinase family. In either case, which of the six hydroxyl groups of myo-inositol is phosphorylated is still unknown. In addition, little is known about the myo-inositol binding mechanism of these enzymes. In this work; we determined two crystal structures: those of the TK2285 protein complexed with the substrates (ATP analogue and myo-inositol) or the reaction products formed by the enzyme. Analysis of the ternary substrates-complex structure and site-directed mutagenesis showed that five residues were involved in the interaction with myo-inositol. Structural comparison with other ribokinase family enzymes indicated that two of the five residues, Q136 and R140, are characteristic of myo-inositol kinase. The crystal structure Of the ternary products-complex, which was prepared by incubating the TK2285 protein with myo-inositol and ATP, holds 1D-myo-inositol 3-phosphate (Ins(3)P) in the active site. NMR and HPLC analyses with a chiral column also indicated that the TK2285 reaction product was Ins(3)P. The results obtained here showed that the TK2285 protein specifically catalyzes the phosphorylation of the 3-OH of myo-inositol. We thus designated TK2285 as myo-inositol 3-kinase (MI3K). The precise identification of the reaction product should provide a sound basis to further explore inositol metabolism in Archaea.