The branched Entner-Doudoroff pathway in Thermoproteus tenax: Enzymes, regulation and physiological function

对嗜热菌中心碳水化合物代谢的一般理解包括在（超）嗜热菌中存在非非磷酸化Entner-Doudoroff（艾德）途径，在嗜盐菌中存在半磷酸化艾德途径。然而，最近结合比较基因组学和生物化学的方法揭示了存在的半磷酸化途径的超嗜热Crenarchaeota Thermoproteus tenax和Sulfolobus solfataricus。最初的生物化学研究导致鉴定了i）一种新型的葡糖酸酯酶，ii）一种双功能2-酮基-3-脱氧-（6-磷酸-）葡糖酸酯（KD（P）G）醛缩酶，iii）第一种古细菌2-酮基-3-脱氧葡糖酸酯（KDG）激酶和iv）一种甘油酸激酶。这些结果表明，在这些生物体中存在两种艾德修饰（半磷酸化和非磷酸化），因此存在分支艾德途径。于T. tenax所提出的分支艾德途径除了充分研究的Embden-Meyerhof-Parnas（EMP）修饰外是有活性的，并且似乎对葡萄糖是特异性的，而它代表了S.太阳神本研究的目的是证实T. tenax，研究其酶，其立体选择性/混杂性，以及该途径的调节和生理功能。为了获得综合的理解，拟议的研究将与结构生物学，系统发育研究以及现代高通量方法（蛋白质组学/磷酸化组学，转录组学）进行不同的合作。该项目不仅有助于了解古细菌的中心碳水化合物代谢及其调控，而且有助于进一步揭示生命所有三个领域的代谢复杂性。

The general understanding of central carbohydrate metabolism in Archaea comprises the presence of the non-nonphosphorylative Entner-Doudoroff (ED) pathway in (hyper)thermophilic Archaea and of the semi-phosphoryltive ED pathway in halophilic Archaea. However, a recent combined comparative genomics and biochemical approach revealed the presence of the semi-phosphorylative pathway in the hyperthermophilic Crenarchaeota Thermoproteus tenax and Sulfolobus solfataricus. Initial biochemical studies led to the identification of i) a novel type of gluconate dehydratase ii) a bifunctional 2-keto-3-deoxy-(6-phospho-)gluconate (KD(P)G) aldolase, iii) the first archaeal 2-keto-3-deoxygluconate (KDG) kinase and iv) a glycerate kinase. These results suggest the presence of both ED modifications (semi- and non-phosphorylative) and thus a branched ED pathway in these organisms. In T. tenax the suggested branched ED pathway is active in addition to the well studied Embden-Meyerhof-Parnas (EMP) modification and seems to be specific for glucose, whereas it represents the only “promiscuous” route for glucose and galactose degradation in S. solfataricus. The goal of this proposed study is to confirm the presence of the branched ED pathway in T. tenax, study its enzymes, their stereoselectivity/promiscuity, as well as the regulation and physiological function of the pathway. To gain an integrated understanding, the proposed studies will be combined in different collaborations with structural biology, phylogenetic studies as well as modern high-throughput approaches (proteomics/phosphorylomics, transcriptomics). This project will not only contribute to the understanding of the archaeal central carbohydrate metabolism and its regulation, but will aid to further unravel the metabolic complexity in all three domains of life.