Theron A, Roth R L, Hoppe H, Parkinson C, van der Westhuyzen C W, Stoychev S, Wiid I, Pietersen R D, Baker B, Kenyon C P
CSIR Biosciences, Pretoria, South Africa.
School of Biomedical Sciences, Charles Sturt University, Orange NSW, Australia.
PLoS One. 2017 Oct 3;12(10):e0185068. doi: 10.1371/journal.pone.0185068. eCollection 2017.
Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.
谷氨酰胺合成酶是氮代谢中一种普遍存在的关键酶，受多达四种调控机制的控制，包括腺苷酸转移酶对十二个亚基中的部分或全部进行腺苷酸化。它被认为是治疗结核病的潜在治疗靶点，对结核分枝杆菌的生长至关重要，并且仅在致病性分枝杆菌菌株的细胞外发现。人谷氨酰胺合成酶不受腺苷酸化机制调控，因此细菌谷氨酰胺合成酶的腺苷酸化形式特别受关注。先前发表的报告表明，当结核分枝杆菌谷氨酰胺合成酶在大肠杆菌中表达时，大肠杆菌腺苷酸转移酶不能最佳地对结核分枝杆菌谷氨酰胺合成酶进行腺苷酸化。在此，我们通过共表达结核分枝杆菌谷氨酰胺合成酶和结核分枝杆菌腺苷酸转移酶，证明了在大肠杆菌中产生可溶性腺苷酸化的结核分枝杆菌谷氨酰胺合成酶。报告了基于ATP的支架抑制剂对腺苷酸化的结核分枝杆菌谷氨酰胺合成酶和去腺苷酸化的结核分枝杆菌谷氨酰胺合成酶的差异抑制作用。基于酶抑制作用选择的化合物在BACTEC 460TB™试验中也显示出抑制结核分枝杆菌的作用，以及在小鼠骨髓来源巨噬细胞试验中对结核分枝杆菌的细胞内抑制作用。
