Structure and function of a metabolic pacemaker in bacterial cell membrane

摘要 糖酵解是真核生物和酵母中保守的最重要的代谢途径之一。 原核生物在该途径中，葡萄糖被分解形成小的3-碳磷酸代谢物， 细胞生长和存活。在微生物中，适当地维持糖酵解对于微生物的发育是重要的。 细菌感染、毒力和抗生素耐药性。在这个项目中，我们的目标是研究结构和 磷脂酰甘油磷酸酶PgpA的功能，以阐明糖酵解的新的调节机制， 细菌。PgpA是一种广泛存在于革兰氏阴性菌中的膜蛋白。我们发现 PgpA作为兼职酶发挥作用;即PgpA不仅参与磷脂生物合成，而且还参与磷脂合成。 通过水解大肠杆菌中关键的3-碳磷酸糖酵解代谢物，作为一种必需的代谢调节剂。 杆菌PgpA在E.大肠杆菌极大地促进了细菌的代谢和生长。我们还 确定了PgpA的一种新的氧化还原调节机制，这对维持细菌代谢很重要 体内平衡我们的研究结果提出了一种假设，即PgpA是一种氧化还原介导的调节机制， 通过控制谷胱甘肽介导的氧化还原平衡调节细菌糖酵解， 代谢信号这种调节机制是新颖的，尚未在任何细胞类型中报道。进一步 了解这种调节机制，我们将研究PgpA如何控制细菌葡萄糖摄取和调节 使用生物化学、微生物学和代谢组学方法的组合来研究糖酵解活性。 了解PgpA如何调节细胞内氧化还原平衡，我们将研究谷胱甘肽的生物合成， 监测PgpA膜表面的氧化还原变化，以证明PgpA如何使用整合的“Ying- 阳”的机制来实现代谢稳态和氧化还原平衡。我们还发现氧化还原介导的 PgpA的调节由PgpA二聚体内的二聚二硫键交联介导。为了获得结构性的见解， 在这一新的氧化还原调节催化机制中，我们将研究PgpA和辅因子的催化活性 使用生物化学测定法在体外响应氧化还原变化的Mg 2+结合。我们还将研究这种分子 使用FRET的机制来证明二聚体交联如何改变蛋白质构象以变构 改变活性位点构象以控制PgpA催化。由于在 PgpA家族，我们将确定PgpA在两个不同的氧化还原（活性/失活）状态下的结构，使用 X射线晶体学和单粒子cryoEM方法，以建立氧化还原的结构基础， PgpA的调节催化机制。这种机制在许多革兰氏阴性病原体中是保守的。我们 研究将揭示理解微生物代谢调节的重要机制。

Abstract Glycolysis constitutes one of the most important metabolic pathways conserved in both eukaryotes and prokaryotes. In the pathway, glucose is broken down to form small 3-carbon phosphate metabolites essential for cell growth and survival. In microorganisms, properly maintaining glycolysis is important for the development of bacterial infection and virulence and antibiotic resistance. In this project, we aim to study the structure and function of phosphatidylglycerol phosphatase PgpA to elucidate a novel regulatory mechanism of glycolysis in bacterium. PgpA is an integral membrane protein ubiquitously found in Gram-negative bacterium. We found that PgpA functions as a moonlighting enzyme; i.e. PgpA is not only involved in phospholipid biosynthesis but also acts as an essential metabolic regulator by hydrolyzing the key 3-carbon phosphate glycolytic metabolites in E. coli. Mutational inactivation of PgpA in E. coli greatly facilitates bacterial metabolism and growth. We have also identified a novel redox-regulatory mechanism of PgpA, which is important to maintain bacterial metabolic homeostasis. Our findings raise the hypothesis for a redox-mediated regulatory mechanism in which PgpA regulates bacterial glycolysis by controlling glutathione-mediated redox balance based on external and internal metabolic signals. This regulatory mechanism is novel and has not yet been reported in any cell type. To further understand this regulatory mechanism, we will study how PgpA controls bacterial glucose uptake and regulate glycolytic activity using a combination of biochemistry, microbiology, and metabolomic approaches.To understand how PgpA regulates intracellular redox balance, we will examine glutathione biosynthesis and monitor redox changes on the membrane surface of PgpA to demonstrate how PgpA uses an integrative “Ying- Yang” mechanism to achieve both metabolic homeostasis and redox balance. We also found the redox-mediated regulation of PgpA is mediated by dimeric disulfide crosslinking within PgpA dimer. To gain structural insights into this novel redox-regulated catalytic mechanism, we will study the catalytic activity of PgpA and co-factor Mg2+ binding in response to redox changes in vitro using biochemical assays. We will also study this molecular mechanism using FRET to demonstrate how dimeric crosslinking alters protein conformation to allosterically change the active site conformation in order to control the PgpA catalysis. Since no structure is available in the PgpA family, we will determine the structures of PgpA in two distinct redox (active/inactivated) states using the X-ray crystallography and single-particle cryoEM approaches to establish a structural basis for the redox- regulated catalytic mechanism of PgpA. This mechanism is conserved in many Gram-negative pathogens. Our studies will reveal an important mechanism to understand metabolic regulation in microorganisms.