Cloning and Expression of the Inositol Monophosphatase Gene from Methanococcus jannaschii and Characterization of the Enzyme
Cloning and Expression of the Inositol Monophosphatase Gene from Methanococcus jannaschii and Characterization of the Enzyme
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詹氏甲烷球菌肌醇单磷酸酶基因的克隆、表达及酶的表征
DOI：
10.1128/aem.64.7.2609-2615.1998
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发表时间：
1998
影响因子：
4.4
通讯作者：
M. Roberts
中科院分区：
文献类型：
--
作者：
Liangjing Chen;M. Roberts
肌醇单磷酸酶（EC 3.1.3.25）在二肌肌醇-1,1 ' -磷酸的生物合成中起着关键作用，二肌肌醇-1,1 ' -磷酸是一种在超嗜热古菌中发现的渗透物。考虑到jannaschi甲烷球菌MJ109基因产物与人肌醇单磷酸酶序列同源性，我们克隆了MJ109基因，在大肠杆菌中表达并检测了MJ109基因的肌醇单磷酸酶活性。纯化后的MJ109基因产物具有肌醇单磷酸酶活性，动力学参数（Km = 0.091±0.016 mM;Vmax = 9.3±0.45 μmol of Pi min−1 mg of protein−1）与哺乳动物和大肠杆菌酶相当。研究了其底物特异性、Mg2+需求、Li+抑制、亚基结合（二聚化）和热稳定性，并与其他肌醇单磷酸酶进行了比较。低浓度Li+和高浓度Mg2+的抑制作用不足，葡萄糖-1-磷酸和对硝基苯基磷酸的水解速率高，是古细菌肌醇单磷酸酶与其他来源的肌醇单磷酸酶最显著的区别。本文从动物肌醇单磷酸酶与人肌醇单磷酸酶的活性位点序列比对和晶体结构等方面探讨了产生这些动力学差异的可能原因。
ABSTRACT Inositol monophosphatase (EC 3.1.3.25 ) plays a pivotal role in the biosynthesis of di-myo-inositol-1,1′-phosphate, an osmolyte found in hyperthermophilic archaea. Given the sequence homology between the MJ109 gene product of Methanococcus jannaschii and human inositol monophosphatase, the MJ109 gene was cloned and expressed in Escherichia coli and examined for inositol monophosphatase activity. The purified MJ109 gene product showed inositol monophosphatase activity with kinetic parameters (Km = 0.091 ± 0.016 mM;Vmax = 9.3 ± 0.45 μmol of Pi min−1 mg of protein−1) comparable to those of mammalian and E. coli enzymes. Its substrate specificity, Mg2+ requirement, Li+inhibition, subunit association (dimerization), and heat stability were studied and compared to those of other inositol monophosphatases. The lack of inhibition by low concentrations of Li+ and high concentrations of Mg2+ and the high rates of hydrolysis of glucose-1-phosphate and p-nitrophenylphosphate are the most pronounced differences between the archaeal inositol monophosphatase and those from other sources. The possible causes of these kinetic differences are discussed, based on the active site sequence alignment between M. jannaschii and human inositol monophosphatase and the crystal structure of the mammalian enzyme.
DOI：
10.1016/s0021-9258(19)70391-3
发表时间：
1980-11
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Loretta M. HallcherS;W. Sherman
通讯作者：
Loretta M. HallcherS;W. Sherman
磷酸肌醇在跨膜信号传导中的作用。
影响因子：
33.6
作者：
Rana,RS;Hokin,LE
通讯作者：
Hokin,LE