The Biology Of Sugar Transport in E Coli

对大肠杆菌糖运输系统的蛋白质组分的结构和调控的研究仍在继续。PTS的第一组分(酶I，EI)被活性部位组氨酸上的磷酸烯醇式丙酮酸(PEP)磷酸化，在需要镁(2+)的反应中生成丙酮酸。与Clore实验室(NIDDK)合作的新研究利用核磁共振技术阐明了大肠杆菌甘露醇转运蛋白细胞质B结构域的三维结构。有序的IIBMtl结构域由一个四链平行的β-折叠组成，一侧有两个螺旋，另一个面有一个额外的螺旋，具有特征的Rossman折叠。活性部位半胱氨酸(Cys-384)是亲核攻击的基础，在磷酸化组氨酸(His-554)的IIAMt1结构域。IIBMtl的结构与蛋白质酪氨酸磷酸酶相似。 与王实验室(Eppley Institute)合作，已经开始对临时秘书处的氮调节部门进行结构分析。IIANtR的X射线结构此前已被其他人报道。为推断IIANtR与NPR形成的络合物的溶液结构奠定了基础，并对其进行了化学位移归属。 葡萄糖IIA的N-末端结构域赋予蛋白质两面性，使其能够穿梭于细胞膜和细胞质之间。研究了与N-末端结构域相对应的合成肽的结构。用核磁共振研究了磷脂和洗涤剂链长对多肽结构和平移扩散系数的影响。对三种阴离子磷脂和四种类脂阴离子洗涤剂进行了评价。在所有情况下，阳离子肽采用两亲性螺旋结构。双链磷脂的链长对多肽构象的影响可以忽略不计，而单链洗涤剂的链长对多肽构象的影响更为显著。短链阴离子磷脂被认为是膜结合肽结构研究中有用的膜仿生模型。 细菌PTS调节多种生理过程，其中一些是由IIAGlc酶介导的。通过表面等离子体共振配基钓鱼，发现了一种新的IIAGlc结合蛋白。该蛋白命名为FRSA(发酵/呼吸开关蛋白)，是YafA基因的47 kDa产物，以前被表示为功能未知。FRSA与未磷酸化的IIAGlc形成1：1的络合物，亲和力高。FRSA的干扰增加了细胞对几种糖的呼吸，同时增加了FRSA的表达，导致了某些糖的发酵速率增加。结果表明，IIAGlc通过与FRSA之间的磷酸化状态依赖的相互作用来感知可用糖种类，从而调节呼吸和发酵之间的通量。

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.