Functional analysis of GlnE, an essential adenylyl transferase in Mycobacterium tuberculosis
Functional analysis of GlnE, an essential adenylyl transferase in Mycobacterium tuberculosis
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DOI：
10.1128/jb.00166-08
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发表时间：
2008-07-01
影响因子：
3.2
通讯作者：
Parish, Tanya
中科院分区：
文献类型：
--
作者：
Carroll, Paul;Pashley, Carey A.;Parish, Tanya
谷氨酰胺合成酶（GS）在氮同化过程中起着重要作用。结核分枝杆菌的主要GS是GlnA 1，这是一种I型GS，其活性受GlnE的转录后修饰控制。GlnE是由调节GS活性的腺苷酰化结构域和去腺苷酰化结构域组成的腺苷酰转移酶。我们先前证明GlnE在M.正常生长培养基中的结核病。在这项研究中，我们进一步表明，GlnE是需要在多种介质条件下，包括在氮限制培养基。我们证明腺苷酸化是M.结核病存活，因为我们能够删除脱氧腺苷化结构域，对生长或GS活性没有明显影响。此外，我们确定了一个关键的天冬氨酸残基在建议的核苷酸转移酶基序。GlnE的温度敏感突变体的产生，并显示在氮限制培养基中的生长和GS活性的缺陷。最后，我们能够在L-甲硫氨酸亚砜亚胺、GS抑制剂和谷氨酰胺补充剂的存在下产生GlnE无效突变体。在这些补充剂的存在下，无效突变体能够与野生型相似地生长。令人惊讶的是，在没有GS抑制的情况下，GlnE突变体能够在液体培养基中存活并生长延长的时间，但在固体培养基上不能。因此，我们已经证实，M的不寻常的要求。结核病的GlnE腺苷酸化活性与细胞中GS的活性有关。
Glutamine synthetase (GS) plays an important role in nitrogen assimilation. The major GS of Mycobacterium tuberculosis is GlnA1, a type I GS whose activity is controlled by posttranscriptional modification by GlnE. GlnE is an adenylyl transferase comprised of an adenylylating domain and a deadenylylating domain which modulate GS activity. We previously demonstrated that GlnE is essential in M. tuberculosis in normal growth medium. In this study, we further show that GlnE is required under multiple medium conditions, including in nitrogen-limited medium. We demonstrate that adenylylation is the critical activity for M. tuberculosis survival, since we were able to delete the deadenylylation domain with no apparent effect on growth or GS activity. Furthermore, we identified a critical aspartate residue in the proposed nucleotidyltransferase motif. Temperature-sensitive mutants of GlnE were generated and shown to have a defect in growth and GS activity in nitrogen-limited medium. Finally, we were able to generate a GlnE null mutant in the presence Of L-methionine sulfoximine, a GS inhibitor, and glutamine supplementation. In the presence of these supplements, the null mutant was able to grow similarly to the wild type. Surprisingly, the GlnE mutant was able to survive and grow for extended periods in liquid medium, but not on solid medium, in the absence of GS inhibition. Thus, we have confirmed that the unusual requirement of M. tuberculosis for GlnE adenylylation activity is linked to the activity of GS in the cell.