The Biology Of Sugar Transport in E Coli

大肠杆菌中糖运输的生物学

• 批准号: 6966841
• 负责人: ALAN PETERKOFSKY
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6966841
• 关键词: Escherichia coli; bacterial genetics; bacterial proteins; carbohydrate transport; enzyme complex; enzyme mechanism; enzyme structure; gene expression; lactose; micelles; microorganism metabolism; nuclear magnetic resonance spectroscopy; phosphodiesterases; phosphorylation; phosphotransferases; protein protein interaction; protein structure function

Structural and regulatory studies on protein components of the E. coli sugar transport system known as the phosphoenolpyruvate:sugar phosphotransferase system (PTS) continued. The first component of the PTS (enzyme I, EI) is phosphorylated by phosphoenolpyruvate (PEP) on an active site histidine in a Mg(2+)-requiring reaction to produce pyruvate. New studies, in collaboration with the Clore laboratory (NIDDK), have elucidated the three-dimensional structure of the cytoplasmic B domain of the Escherichia coli mannitol transporter using NMR. The ordered IIBMtl domain consists of a four-stranded parallel beta-sheet flanked by two helices on one face and an additional helix on the opposite face with a characteristic Rossman fold. The active site cysteine (Cys-384) is primed for nucleophilic attack at the phosphorylated histidine (His-554) of the IIAMtl domain. The structure of IIBMtl is similar to that of protein tyrosine phosphatase. In collaboration with the Wang laboratory (Eppley Institute), structural analysis of the nitrogen regulatory arm of the PTS has been started. The X-ray structure of IIANtr has previously been reported by others. To lay the groundwork for the deduction of the solution structure of the complex of IIANtr with NPr, the chemical shift assignments for IIANtr have been made. The N-terminal domain of glucose IIA confers amphitropism to the protein, allowing it to shuttle between the membrane and cytoplasm. The structure of a synthetic peptide corresponding to the N-terminal domain was studied. The effects of phospholipids or detergent chain length on the structure and translational diffusion coefficient of the peptide were investigated by NMR. Three anionic phospholipids and four lipid-mimicking anionic detergents were evaluated. In all cases, the cationic peptide adopts an amphipathic helical structure. While the chain-length of the two-chain phospholipids has a negligible effect on the peptide conformation, the effect of chain length of single-chain detergents is more significant. Short-chain anionic phospholipids are proposed to be useful membrane-mimetic models for the structural elucidation of membrane-binding peptides. The bacterial PTS regulates a variety of physiological processes, some of which are mediated by Enzyme IIAGlc. A novel IIAGlc binding protein was discovered by ligand fishing using surface plasmon resonance. The protein, named FrsA (for fermentation/respiration switch protein), is the 47 kDa product of the yafA gene, previously denoted as ?function unknown?. FrsA forms a 1:1 complex with unphosphorylated IIAGlc with a high affinity. Disruption of frsA increased cellular respiration on several sugars, while increased FrsA expression resulted in an increased fermentation rate of some sugars. The results are interpreted to indicate that IIAGlc regulates the flux between respiration and fermentation by sensing the available sugar species via a phosphorylation state dependent interaction with FrsA.

对大肠杆菌的蛋白质组分进行了结构和调控研究。大肠杆菌糖转运系统称为磷酸烯醇丙酮酸：糖磷酸转移酶系统（PTS）继续。PTS的第一组分（酶I，EI）在需要Mg（2+）的反应中被活性位点组氨酸上的磷酸烯醇丙酮酸（PEP）磷酸化以产生丙酮酸。与Clore实验室（NIDDK）合作的新研究已经使用NMR阐明了大肠杆菌甘露醇转运蛋白胞质B结构域的三维结构。有序的IIBMt 1结构域由四链平行β-折叠组成，在一个面上侧接两个螺旋，在相对面上侧接具有特征性Rossman折叠的另外的螺旋。活性位点半胱氨酸（Cys-384）在IIAMt 1结构域的磷酸化组氨酸（His-554）处引发亲核攻击。IIBMt 1的结构与蛋白酪氨酸磷酸酶的结构相似。 与Wang实验室（Eppley研究所）合作，已开始对临时秘书处的氮调节臂进行结构分析。IIANtr的X射线结构先前已被其他人报道。对IIANtr的化学位移进行了归属，为进一步推导IIANtr与NPr配合物的溶液结构奠定了基础。 葡萄糖IIA的N-末端结构域赋予蛋白质两性性，使其能够在膜和细胞质之间穿梭。研究了与N-末端结构域对应的合成肽的结构。通过核磁共振研究了磷脂或洗涤剂链长对肽的结构和平移扩散系数的影响。评估了三种阴离子磷脂和四种模拟脂质的阴离子洗涤剂。在所有情况下，阳离子肽采用两亲性螺旋结构。虽然双链磷脂的链长对肽构象的影响可以忽略不计，但单链洗涤剂的链长的影响更为显著。短链阴离子磷脂被认为是膜结合肽结构解析的有用的膜模拟模型。 细菌PTS调节多种生理过程，其中一些由酶IIAGlc介导。利用表面等离子体共振通过配体钓鱼发现了一种新的IIAGlc结合蛋白。该蛋白质，命名为FrsA（发酵/呼吸开关蛋白），是47 kDa的产品的yafA基因，以前表示为？功能未知？FrsA与未磷酸化的IIAGlc形成高亲和力的1：1复合物。干扰的FrsA增加细胞呼吸几种糖，而增加FrsA的表达导致一些糖的发酵速率增加。结果被解释为指示IIAGlc通过经由与FrsA的磷酸化状态依赖性相互作用感测可用糖种类来调节呼吸和发酵之间的通量。