IN VIVO & IN VITRO NMR STUDIES OF METHANOGEN METABOLISM

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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.