Oxidative stress inactivates cobalamin-independent methionine synthase (MetE) in Escherichia coli.
Oxidative stress inactivates cobalamin-independent methionine synthase (MetE) in Escherichia coli.
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DOI：
10.1371/journal.pbio.0020336
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发表时间：
2004-11
期刊：
影响因子：
9.8
通讯作者：
Matthews RG
中科院分区：
文献类型：
--
作者：
Hondorp ER;Matthews RG
在自然界中，大肠杆菌暴露在恶劣和非理想的生长环境中——营养可能是有限的，细胞经常受到氧化应激的挑战。对于面临这些现实的大肠杆菌细胞来说，氧化应激、蛋氨酸可用性和催化蛋氨酸生物合成最后一步的酶——钴胺非依赖性蛋氨酸合成酶（MetE）之间似乎存在联系。我们发现，大肠杆菌细胞在最小培养基中生长过程中遭受短暂氧化应激会产生蛋氨酸营养不良，这可以追溯到对MetE的影响。进一步的实验表明，纯化酶被氧化谷胱甘肽（GSSG）以与蛋白质氧化相关的速率灭活。通过对每个还原半胱氨酸的n端选择性切割，确定了独特的氧化位点，并通过液相色谱-质谱分析了结果。GSSG对MetE的化学计量谷胱甘肽化发生在半胱氨酸645上，它战略性地位于活性位点的入口。在两种含有高度氧化细胞质环境的不同大肠杆菌菌株中进行的硫醇捕获实验获得了MetE在体内氧化的直接证据。此外，MetE在巯基氧化剂二胺处理的野生型大肠杆菌中被完全氧化；还原酶在细胞恢复正常生长之前再次出现。我们认为，对于在最小培养基中经历氧化条件的大肠杆菌，MetE很容易失活，导致细胞蛋氨酸限制。蛋白质的谷胱甘肽化提供了一种策略来调节酶的体内活性，同时以一种容易可逆的方式保护活性部位免受进一步损害。虽然蛋白质的谷胱甘肽化是真核生物中相当常见的氧化还原调节模式，但已知大肠杆菌中很少有蛋白质以这种方式进行修饰。我们的结果是补充了Leichert和Jakob在随附论文中提出的独立发现，这提供了证据，证明MetE是大肠杆菌中最容易氧化的蛋白质之一。在真核生物中，参与蛋白质合成的关键蛋白谷胱甘肽化导致翻译抑制。我们的研究表明，大肠杆菌采用了一种更简单的机制来达到同样的效果。在氧化应激条件下，大肠杆菌中的蛋氨酸营养不良是催化蛋氨酸生物合成最后一步（MetE）的酶谷胱甘肽化的结果。
In nature, Escherichia coli are exposed to harsh and non-ideal growth environments—nutrients may be limiting, and cells are often challenged by oxidative stress. For E. coli cells confronting these realities, there appears to be a link between oxidative stress, methionine availability, and the enzyme that catalyzes the final step of methionine biosynthesis, cobalamin-independent methionine synthase (MetE). We found that E. coli cells subjected to transient oxidative stress during growth in minimal medium develop a methionine auxotrophy, which can be traced to an effect on MetE. Further experiments demonstrated that the purified enzyme is inactivated by oxidized glutathione (GSSG) at a rate that correlates with protein oxidation. The unique site of oxidation was identified by selectively cleaving N-terminally to each reduced cysteine and analyzing the results by liquid chromatography mass spectrometry. Stoichiometric glutathionylation of MetE by GSSG occurs at cysteine 645, which is strategically located at the entrance to the active site. Direct evidence of MetE oxidation in vivo was obtained from thiol-trapping experiments in two different E. coli strains that contain highly oxidizing cytoplasmic environments. Moreover, MetE is completely oxidized in wild-type E. coli treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. We argue that for E. coli experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein provides a strategy to modulate in vivo activity of the enzyme while protecting the active site from further damage, in an easily reversible manner. While glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in E. coli are known to be modified in this manner. Our results are complementary to the independent findings of Leichert and Jakob presented in the accompanying paper, which provide evidence that MetE is one of the proteins in E. coli most susceptible to oxidation. In eukaryotes, glutathionylation of key proteins involved in protein synthesis leads to inhibition of translation. Our studies suggest a simpler mechanism is employed by E. coli to achieve the same effect. Under conditions of oxidative stress, methionine auxotrophy in E. coli is a result of glutathionylation of the enzyme that catalyzes the final step of methionine biosynthesis (MetE)
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