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

对古生代中枢性碳水化合物代谢的一般理解包括在(超)嗜热古生代中存在非磷酸化的Entner-Doudoroff(ED)途径和在嗜盐古生代中存在半磷酸化的ED途径。然而，最近的比较基因组学和生物化学相结合的方法揭示了在高温嗜热的Crenarota Thermoprotus tenax和Sulfolobus solfararicus中存在半磷酸化途径。初步的生化研究结果表明：1)一种新型的葡萄糖酸脱水酶，2)一种双功能的2-酮-3-脱氧-(6-磷酸)葡萄糖酸(KD(P)G)缩醛酶，三)第一个古生型2-酮-3-脱氧葡萄糖酸(KDG)激酶和一种甘油酸激酶。这些结果表明，在这些生物体中既存在ED修饰(半磷酸化和非磷酸化)，因此存在分支的ED途径。在T.tenax中，所建议的分支ED途径除了被广泛研究的Embden-Meyerhof-Parns(EMP)修饰外，还是活跃的，并且似乎是葡萄糖的专一性途径，而它代表了在S.solfararicus中葡萄糖和半乳糖降解的唯一“混杂”途径。本研究的目的是证实T.tenax中存在分支ED途径，研究其酶类、立体选择性/混杂，以及该途径的调节和生理功能。为了获得全面的理解，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生代中枢碳水化合物代谢及其调控，而且有助于进一步揭开生命三个领域中代谢的复杂性。

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.