Mehta Ranjana, Pearson Josh T, Mahajan Sumit, Nath Abhinav, Hickey Mark J, Sherman David R, Atkins William M
Departments of Pathobiology and Medicinal Chemistry, University of Washington, Seattle, Washington 98195-7610, USA.
J Biol Chem. 2004 May 21;279(21):22477-82. doi: 10.1074/jbc.M401652200. Epub 2004 Mar 22.
Bacterial glutamine synthetases (GSs) are complex dodecameric oligomers that play a critical role in nitrogen metabolism, converting ammonia and glutamate to glutamine. Recently published reports suggest that GS from Mycobacterium tuberculosis (MTb) may be a therapeutic target (Harth, G., and Horwitz, M. A. (2003) Infect. Immun. 71, 456-464). In some bacteria, GS is regulated via adenylylation of some or all of the subunits within the aggregate; catalytic activity is inversely proportional to the extent of adenylylation. The adenylylation and deadenylylation of GS are catalyzed by adenylyl transferase (ATase). Here, we demonstrate via electrospray ionization mass spectrometry that GS from pathogenic M. tuberculosis is adenylylated by the Escherichia coli ATase. The adenylyl group can be hydrolyzed by snake venom phosphodiesterase to afford the unmodified enzyme. The site of adenylylation of MTb GS by the E. coli ATase is Tyr-406, as indicated by the lack of adenylylation of the Y406F mutant, and, as expected, is based on amino acid sequence alignments. Using electrospray ionization mass spectroscopy methodology, we found that GS is not adenylylated when obtained directly from MTb cultures that are not supplemented with glutamine. Under these conditions, the highly related but non-pathogenic Mycobacterium bovis BCG yields partially ( approximately 25%) adenylylated enzyme. Upon the addition of glutamine to the cultures, the MTb GS becomes significantly adenylylated ( approximately 30%), whereas the adenylylation of M. bovis BCG GS does not change. Collectively, the results demonstrate that MTb GS is a substrate for E. coli ATase, but only low adenylylation states are accessible. This parallels the low adenylylation states observed for GS from mycobacteria and suggests the intriguing possibility that adenylylation in the pathogenic versus non-pathogenic mycobacteria is differentially regulated.
细菌谷氨酰胺合成酶（GSs）是复杂的十二聚体寡聚体，在氮代谢中起关键作用，将氨和谷氨酸转化为谷氨酰胺。最近发表的报告表明，结核分枝杆菌（MTb）的GS可能是一个治疗靶点（Harth, G., and Horwitz, M. A. (2003) Infect. Immun. 71, 456 - 464）。在一些细菌中，GS通过聚集体内部分或全部亚基的腺苷酸化进行调节；催化活性与腺苷酸化程度成反比。GS的腺苷酸化和去腺苷酸化由腺苷酸转移酶（ATase）催化。在此，我们通过电喷雾电离质谱证明，致病性结核分枝杆菌的GS被大肠杆菌ATase腺苷酸化。腺苷基团可被蛇毒磷酸二酯酶水解，得到未修饰的酶。大肠杆菌ATase对结核分枝杆菌GS的腺苷酸化位点是Tyr-406，Y406F突变体缺乏腺苷酸化表明了这一点，并且正如预期的那样，这是基于氨基酸序列比对得出的。使用电喷雾电离质谱方法，我们发现直接从未添加谷氨酰胺的结核分枝杆菌培养物中获得的GS未被腺苷酸化。在这些条件下，高度相关但无致病性的牛分枝杆菌卡介苗（M. bovis BCG）产生部分（约25%）腺苷酸化的酶。向培养物中添加谷氨酰胺后，结核分枝杆菌GS显著腺苷酸化（约30%），而牛分枝杆菌卡介苗GS的腺苷酸化没有变化。总体而言，结果表明结核分枝杆菌GS是大肠杆菌ATase的底物，但只能达到低腺苷酸化状态。这与分枝杆菌GS观察到的低腺苷酸化状态相似，并暗示了致病性与非致病性分枝杆菌中腺苷酸化受到差异调节的有趣可能性。
