IN VIVO & IN VITRO NMR STUDIES OF METHANOGEN METABOLISM

产甲烷菌是一种挑剔的厌氧菌，其新陈代谢集中在 将二氧化碳还原为甲烷。这些独特的生物已被归类为 具有与进化上截然不同的特征的“古细菌” 原核生物和真核生物。这些细胞代谢的许多方面 生物体的情况尚不清楚。在这项建议中，体内和体外核磁共振 光谱学将被用来解开碳和氮的同化和 热自养甲烷杆菌的生物能量学特性 菌株DeltaH。具体地说，我们将(I)阐明 2，3-环吡磷酸甘油酸酯(CPP)，主要的可溶性碳和 利用~(13)C核磁共振研究糖异生中的磷库；(Ii)纯化和 表征合成和降解CPP的酶；(Iii)检测 独特的二氧化碳固定途径，导致不对称标记 乙酰辅酶A(使用13C核磁共振)；(Iv)进一步表征氮 使用15N核磁共振进行同化；以及(V)使用31P监测细胞生物能量学 核磁共振。这项工作的结果应该有助于更好地理解 碳水化合物合成的新中间体CPP的代谢作用 碳和氮的同化是如何在这种产甲烷菌中被调节的， 并将代谢途径与其他更常见的生物体进行比较。

Methanogens are fastidious anaerobes whose metabolism centers around the reduction of CO2 to CH4. These unique organisms have been classified a "archaebacteria" with characteristics evolutionarily distinct from procaryotes and eukaryotes. Many aspects of cellular metabolism in these organisms are unclear. In this proposal in vivo and in vitro NMR spectroscopy will be used to unravel carbon and nitrogen assimilation and specific aspects of bioenergetics in Methanobacterium thermoautotrophicum strain DeltaH. Specifically, we will (i) elucidate the exact role of 2,3-Cyclopyrophospho-glycerate (CPP), the major soluble carbon and phosphorus pool, in gluconeogenesis using 13C NMR; (ii) purify and characterize the enzymes that synthesize and degrade CPP; (iii) examine the unique CO2 fixation pathway that gives rise to asymmetric labeling of acetyl coenzyme A (using 13C NMR); (iv) further characterize nitrogen assimilation using 15N NMR; and (v) monitor cell bioenergetics with 31p NMR. The results of this work should lead to a better understanding of the metabolic role of CPP, (a novel intermediate in carbohydrate synthesis) and of how carbon and nitrogen assimilation are regulated in this methanogen, and to a comparison of metabolic pathways with other more common organisms.