Human 1-D-myo-inositol-3-phosphate synthase is functional in yeast
Human 1-D-myo-inositol-3-phosphate synthase is functional in yeast
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DOI：
10.1074/jbc.m312078200
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发表时间：
2004-05-21
影响因子：
4.8
通讯作者：
Greenberg, ML
中科院分区：
文献类型：
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作者：
Ju, SL;Shaltiel, G;Greenberg, ML
我们克隆、测序并表达了编码1- D-肌肌醇-3-磷酸（MIP）合酶（hINO 1）的人cDNA.编码的62- kDa人类酶将D-葡萄糖6-磷酸转化为1- D-肌-肌醇3-磷酸，这是肌醇从头生物合成的限速步骤。从大肠杆菌中纯化的重组人MIP合酶的活性最适pH为8.0，37 ℃，对葡萄糖6-磷酸和NAD（+）的Km值分别为0.57 mM和8 μ M。NH 4+和K+是较好的激活剂，而Na+、Li+、Mg ~（2+）、Mn ~（2+）对酶活性有较强的抑制作用。在缺乏MIP合酶的酵母ino 1Delta突变体（ino 1Delta/hINO 1）中表达的蛋白质补充了突变体的肌醇营养缺陷型，并导致肌醇分泌。MIP合酶活性和细胞内肌醇分别降低约35%和25%，当ino 1Delta/hINO 1在治疗相关浓度的抗双相药物丙戊酸盐（0.6 mM）存在下生长时。然而，在该浓度下，丙戊酸盐未抑制纯化MIP合酶的体外活性，表明药物的抑制作用是间接的。由于肌醇代谢可能在双相情感障碍的病因学和治疗中发挥关键作用，因此肌醇生物合成中关键酶的功能保护强调了酵母模型在这种疾病研究中的作用。
We have cloned, sequenced, and expressed a human cDNA encoding 1- D- myo- inositol- 3- phosphate ( MIP) synthase ( hINO1). The encoded 62- kDa human enzyme converted D- glucose 6- phosphate to 1- D- myo- inositol 3- phosphate, the rate- limiting step for de novo inositol biosynthesis. Activity of the recombinant human MIP synthase purified from Escherichia coli was optimal at pH 8.0 at 37degreesC and exhibited K-m values of 0.57 mM and 8 muM for glucose 6- phosphate and NAD(+), respectively. NH4+ and K+ were better activators than other cations tested ( Na+, Li+, Mg2+, Mn2+), and Zn2+ strongly inhibited activity. Expression of the protein in the yeast ino1Delta mutant lacking MIP synthase ( ino1Delta/ hINO1) complemented the inositol auxotrophy of the mutant and led to inositol excretion. MIP synthase activity and intracellular inositol were decreased about 35 and 25%, respectively, when ino1Delta/ hINO1 was grown in the presence of a therapeutically relevant concentration of the anti- bipolar drug valproate ( 0.6 mM). However, in vitro activity of purified MIP synthase was not inhibited by valproate at this concentration, suggesting that inhibition by the drug is indirect. Because inositol metabolism may play a key role in the etiology and treatment of bipolar illness, functional conservation of the key enzyme in inositol biosynthesis underscores the power of the yeast model in studies of this disorder.