Institute of Microbiology, ETH Zurich, 8093 Zurich, Switzerland.
Université de Toulouse, Institut National des Sciences Appliquées, Université Paul Sabatier, Institut National Polytechnique de Toulouse, Laboratoire D'Ingénierie des Systèmes Biologiques et des Procédés, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
J Biol Chem. 2012 Jan 2;287(1):757-766. doi: 10.1074/jbc.M111.305219. Epub 2011 Nov 21.
Acetyl-CoA assimilation was extensively studied in organisms harboring the glyoxylate cycle. In this study, we analyzed the metabolism of the facultative methylotroph Methylobacterium extorquens AM1, which lacks isocitrate lyase, the key enzyme in the glyoxylate cycle, during growth on acetate. MS/MS-based proteomic analysis revealed that the protein repertoire of M. extorquens AM1 grown on acetate is similar to that of cells grown on methanol and includes enzymes of the ethylmalonyl-CoA (EMC) pathway that were recently shown to operate during growth on methanol. Dynamic 13C labeling experiments indicate the presence of distinct entry points for acetate: the EMC pathway and the TCA cycle. 13C steady-state metabolic flux analysis showed that oxidation of acetyl-CoA occurs predominantly via the TCA cycle and that assimilation occurs via the EMC pathway. Furthermore, acetyl-CoA condenses with the EMC pathway product glyoxylate, resulting in malate formation. The latter, also formed by the TCA cycle, is converted to phosphoglycerate by a reaction sequence that is reversed with respect to the serine cycle. Thus, the results obtained in this study reveal the utilization of common pathways during the growth of M. extorquens AM1 on C1 and C2 compounds, but with a major redirection of flux within the central metabolism. Furthermore, our results indicate that the metabolic flux distribution is highly complex in this model methylotroph during growth on acetate and is fundamentally different from organisms using the glyoxylate cycle.
乙酰辅酶 A 的同化作用在具有乙醛酸循环的生物中得到了广泛的研究。在这项研究中，我们分析了兼性甲基营养菌 Methylobacterium extorquens AM1 的代谢，该菌缺乏异柠檬酸裂解酶，这是乙醛酸循环中的关键酶，在乙酸盐生长时。基于 MS/MS 的蛋白质组学分析表明，在乙酸盐上生长的 M. extorquens AM1 的蛋白质谱与在甲醇上生长的细胞相似，并且包括乙基丙二酸辅酶 A (EMC) 途径的酶，最近在甲醇生长期间发现该途径起作用。动态 13C 标记实验表明，乙酸盐有不同的进入点：EMC 途径和 TCA 循环。13C 稳态代谢通量分析表明，乙酰辅酶 A 的氧化主要通过 TCA 循环发生，而同化作用通过 EMC 途径发生。此外，乙酰辅酶 A 与 EMC 途径产物乙醛酸缩合，导致苹果酸的形成。后者也由 TCA 循环形成，通过与丝氨酸循环相反的反应序列转化为磷酸甘油酸。因此，本研究的结果揭示了在 M. extorquens AM1 利用 C1 和 C2 化合物生长过程中共同途径的利用，但在中心代谢中主要是通量的重新定向。此外，我们的结果表明，在乙酸盐生长过程中，这种模型甲基营养菌的代谢通量分布非常复杂，与利用乙醛酸循环的生物体根本不同。
