In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum
In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum
复制标题
DOI：
10.1074/jbc.m908728199
复制
发表时间：
2000-11-17
影响因子：
4.8
通讯作者：
Sahm, H
中科院分区：
文献类型：
--
作者：
Petersen, S;de Graaf, AA;Sahm, H
在许多微生物的回补节点处，C - 3 - C - 4代谢物的相互转化涉及一系列复杂的反应。C - 3羧化成草酰乙酸可源于磷酸烯醇式丙酮酸和丙酮酸，同时可能存在多种C - 4脱羧酶。这种平行反应的功能尚未完全被理解。我们在此利用一种基于13C - 核磁共振的策略，对谷氨酸棒杆菌回补节点处的各个通量进行定量，谷氨酸棒杆菌是一种具有多种羧化和脱羧反应的细菌实例。谷氨酸棒杆菌以13C标记的葡萄糖同位素异构体混合物作为主要碳源以及13C标记的乳酸作为共底物进行培养。对58种同位素异构体以及生物质化合物的15种位置标记进行了定量。应用一种普遍适用的数学模型，该模型包含代谢物质量和碳标记平衡，结果表明丙酮酸羧化酶对C - 3羧化的贡献率为91 ± 7%。体内总羧化速率为1.28 ± 0.14毫摩尔/克干重/小时，比生物合成对羧化代谢物的需求高出3倍。过量的草酰乙酸通过磷酸烯醇式丙酮酸羧激酶再循环为磷酸烯醇式丙酮酸。这表明回补节点处的反应可能除了仅为生物合成提供C - 4代谢物之外，还具有其他作用。
The C-3-C-4 metabolite interconversion at the anaplerotic node in many microorganisms involves a complex set of reactions. C-3, carboxylation to oxaloacetate can originate from phosphoenolpyruvate and pyruvate, and at the Same time multiple C-4-decarboxylating enzymes may be present. The functions of such parallel reactions are not yet fully understood. Using a C-13 NMR-based strategy, we here quantify the individual fluxes at the anaplerotic node of Corynebacterium glutamicum, which is an example of a bacterium possessing multiple carboxylation and decarboxylation reactions. C. glutamicum was grown with a C-13-labeled glucose isotopomer mixture as the main carbon source and 13C-labeled lactate as a cosubstrate. 58 isotopomers as well as 15 positional labels of biomass compounds were quantified. Applying a generally applicable mathematical model to include metabolite mass and carbon labeling balances, it is shown that pyruvate carboxylase contributed 91 +/- 7% to C-3 carboxylation. The total in vivo carboxylation rate of 1.28 +/- 0.14 mmol/g dry weight/h exceeds the demand of carboxylated metabolites for biosyntheses 3-fold. Excess oxaloacetate was recycled to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase. This shows that the reactions at the anaplerotic node might serve additional purposes other than only providing C-4 metabolites for biosynthesis.