13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation
13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation
复制标题
13C 代谢通量分析确定了分枝杆菌中丙酮酸异化的不寻常途径，需要异柠檬酸裂解酶和二氧化碳固定
作者：
D. Beste;B. Bonde;Nathaniel D. Hawkins;J. Ward;M. H. Beale;S. Noack;Katharina Nö;N. Kruger;R. Ratcliffe;J. McFadden;Eric J. Rubin
结核分枝杆菌需要异柠檬酸裂解酶（ICL）在体内生长和毒力。结核分枝杆菌在营养匮乏时也需要ICL来生存，并在甘油限制趋化器的稳态生长中发挥作用，这表明该酶的功能超出了脂肪代谢。由于异柠檬酸裂解酶是一种潜在的药物靶点，阐明该酶的作用具有重要意义；然而，异柠檬酸裂解酶的作用从未在体内通量水平上进行过研究。本研究表明，两个icl基因中的一个缺失会损害牛分枝杆菌BCG在碳限制趋化器中缓慢生长的复制。为了进一步了解异柠檬酸裂解酶在分枝杆菌中心代谢中的作用，首次采用13c代谢通量分析（MFA）研究了生长速率对其体内通量的影响。示踪实验在卡介苗或结核分枝杆菌的稳态趋化培养物中进行，并提供13C标记的甘油或碳酸氢钠。通过测量蛋白质衍生氨基酸中的13C同位素标记模式和酶活性分析，我们已经确定了丙酮酸异化的新途径的活性。我们将其命名为GAS途径，因为它利用乙醛酸分流和异氧性反应氧化丙酮酸，并利用琥珀酰辅酶a合成酶生成琥珀酰辅酶a，同时通过三羧酸循环的琥珀酸-草酰乙酸段的通量非常低。我们证实结核分枝杆菌可以将二氧化碳中的碳固定到生物质中。由于人类宿主体内含有丰富的二氧化碳，这一发现需要在体内进一步研究，因为二氧化碳固定可能提供一个可用于新药的易感性点。本研究也为进一步利用13C-MFA研究结核分枝杆菌的代谢提供了平台。
Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL) for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using 13C-metabolic flux analysis (MFA). Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with 13C labeled glycerol or sodium bicarbonate. Through measurements of the 13C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate – oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO2 into biomass. As the human host is abundant in CO2 this finding requires further investigation in vivo as CO2 fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using 13C-MFA.
DOI：
10.1073/pnas.0501605102
发表时间：
2005-07-26
影响因子：
11.1
作者：
Tian, J;Bryk, R;Nathan, C
通讯作者：
Nathan, C
影响因子：
--
作者：
de Carvalho LP;Zhao H;Dickinson CE;Arango NM;Lima CD;Fischer SM;Ouerfelli O;Nathan C;Rhee KY
通讯作者：
Rhee KY