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首页> 外文期刊>Biochemistry >Purification and biochemical characterization of Mycobacterium tuberculosis SuhB, an inositol monophosphatase involved in inositol biosynthesis.
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Purification and biochemical characterization of Mycobacterium tuberculosis SuhB, an inositol monophosphatase involved in inositol biosynthesis.

机译：结核分枝杆菌SuhB（一种参与肌醇生物合成的肌醇单磷酸酶）的纯化和生化特性。

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Phosphatidylinositol is an essential component of mycobacteria, and phosphatidylinositol-based lipids such as phosphatidylinositolmannosides, lipomannan, and lipoarabinomannan are major immunomodulatory components of the Mycobacterium tuberculosis cell wall. Inositol monophosphatase (EC 3.1.3.25) is a crucial enzyme in the biosynthesis of free myo-inositol from inositol-1-phosphate, a key substrate for the phosphatidylinositol synthase in mycobacteria. Analysis of the M. tuberculosis genome suggested the presence of four M. tuberculosis gene products that exhibit an inositol monophosphatase signature. In the present report, we have focused on SuhB, which possesses the highest degree of homology with human inositol monophosphatase. SuhB gene was cloned into an E. coli expression vector to over-produce a His-tagged protein, which was purified and characterized. SuhB required divalent metal ions for functional inositol monophosphatase activity, with Mg(2+) being the strongest activator. Inositol monophosphatase activity catalyzed by SuhB was inhibited by the monovalent cation lithium (IC(50) = 0.9 mM). As anticipated, inositol-1-phosphate was the preferred substrate (K(m) = 0.177 +/- 0.025 mM; k(cat) = 3.6 +/- 0.2 s(-)(1)); however, SuhB was also able to hydrolyze a variety of polyol phosphates such as glucitol-6-phosphate, glycerol-2-phosphate, and 2'-AMP. To provide further insight into the structure-function relationship of SuhB, different mutant proteins were generated (E83D, D104N, D107N, W234L, and D235N). These mutations almost completely abrogated inositol monophosphatase activity, thus underlining the importance of these residues in inositol-1-phosphate dephosphorylation. We also identified L81 as a key residue involved in sensitivity to lithium. The L81A mutation rendered SuhB inositol monophosphatase activity 10-fold more resistant to inhibition by lithium (IC(50) = 10 mM). These studies provide the first steps in the delineation of the biosynthesis of the key metabolite inositol in M. tuberculosis.
机译：磷脂酰肌醇是分枝杆菌的基本组成部分，而基于磷脂酰肌醇的脂质如磷脂酰肌醇甘露糖苷，脂质甘露聚糖和脂质阿拉伯糖甘露聚糖是结核分枝杆菌细胞壁的主要免疫调节成分。肌醇单磷酸酶（EC 3.1.3.25）是从肌醇-1-磷酸生物合成游离肌醇中的关键酶，肌醇-1-磷酸是分枝杆菌中磷脂酰肌醇合成酶的关键底物。结核分枝杆菌基因组的分析表明存在四个显示肌醇单磷酸酶签名的结核分枝杆菌基因产物。在本报告中，我们集中于SuhB，它与人肌醇单磷酸酶具有最高程度的同源性。将SuhB基因克隆到大肠杆菌表达载体中以过量产生His-tagged蛋白，然后对其进行纯化和鉴定。 SuhB需要二价金属离子才能发挥功能性肌醇单磷酸酶活性，其中Mg（2+）是最强的活化剂。 SuhB催化的肌醇单磷酸酶活性被单价阳离子锂（IC（50）= 0.9 mM）抑制。如所预期的，肌醇-1-磷酸是优选的底物（K（m）＝ 0.177 +/- 0.025mM; k（cat）＝ 3.6 +/- 0.2s（-）（1））;和然而，SuhB也能够水解多种多元醇磷酸酯，例如6-磷酸葡萄糖醇，-2-磷酸甘油和2'-AMP。为了进一步了解SuhB的结构-功能关系，生成了不同的突变蛋白（E83D，D104N，D107N，W234L和D235N）。这些突变几乎完全消除了肌醇单磷酸酶的活性，因此强调了这些残基在肌醇-1-磷酸去磷酸化中的重要性。我们还确定了L81是对锂敏感的关键残基。 L81A突变使SuhB肌醇单磷酸酶活性对锂的抑制能力提高了10倍（IC（50）= 10 mM）。这些研究为描述结核分枝杆菌中关键代谢物肌醇的生物合成提供了第一步。

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