O'Hare Helen M, Durán Rosario, Cerveñansky Carlos, Bellinzoni Marco, Wehenkel Anne Marie, Pritsch Otto, Obal Gonzalo, Baumgartner Jens, Vialaret Jérome, Johnsson Kai, Alzari Pedro M
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
Mol Microbiol. 2008 Dec;70(6):1408-23. doi: 10.1111/j.1365-2958.2008.06489.x. Epub 2008 Oct 17.
Protein kinase G of Mycobacterium tuberculosis has been implicated in virulence and in regulation of glutamate metabolism. Here we show that this kinase undergoes a pattern of autophosphorylation that is distinct from that of other M. tuberculosis protein kinases characterized to date and we identify GarA as a substrate for phosphorylation by PknG. Autophosphorylation of PknG has little effect on kinase activity but promotes binding to GarA, an interaction that is also detected in living mycobacteria. PknG phosphorylates GarA at threonine 21, adjacent to the residue phosphorylated by PknB (T22), and these two phosphorylation events are mutually exclusive. Like the homologue OdhI from Corynebacterium glutamicum, the unphosphorylated form of GarA is shown to inhibit alpha-ketoglutarate decarboxylase in the TCA cycle. Additionally GarA is found to bind and modulate the activity of a large NAD(+)-specific glutamate dehydrogenase with an unusually low affinity for glutamate. Previous reports of a defect in glutamate metabolism caused by pknG deletion may thus be explained by the effect of unphosphorylated GarA on these two enzyme activities, which may also contribute to the attenuation of virulence.
结核分枝杆菌的蛋白激酶G与毒力及谷氨酸代谢调节有关。在此我们表明，该激酶经历一种自磷酸化模式，这与迄今已鉴定的其他结核分枝杆菌蛋白激酶不同，并且我们鉴定出GarA是PknG磷酸化的底物。PknG的自磷酸化对激酶活性影响很小，但促进与GarA的结合，这种相互作用在活的分枝杆菌中也能检测到。PknG在苏氨酸21处磷酸化GarA，该位点与被PknB磷酸化的残基（T22）相邻，并且这两个磷酸化事件相互排斥。与谷氨酸棒杆菌的同源物OdhI一样，未磷酸化形式的GarA被证明可抑制三羧酸循环中的α-酮戊二酸脱羧酶。此外，发现GarA可结合并调节一种对谷氨酸具有异常低亲和力的大型NAD⁺特异性谷氨酸脱氢酶的活性。因此，先前关于pknG缺失导致谷氨酸代谢缺陷的报道可能可以通过未磷酸化的GarA对这两种酶活性的影响来解释，这也可能导致毒力减弱。
