Characterization of bacillosamine synthesis in C. jejuni

描述（由申请人提供）：由于涉及的反应物和产物的双峰性质，发生在膜-细胞溶胶界面的过程的生化表征本质上是具有挑战性的。从革兰氏阴性肠病原菌空肠弯曲杆菌中合成杆菌胺是一个具有代表性的生化分析过程。在空肠杆菌中，杆菌胺是七聚体的第一个碳水化合物，聚在细胞质中，通过膜运输，并在质周空间与新生蛋白n连接。已克隆出参与杆菌胺生物合成的酶的编码位点，并鉴定出七聚体聚糖。然而，在多糖生物合成过程中，质膜、酶的跨膜结构域和膜结合的焦磷酸杆菌烯醇分子的作用尚不清楚。在本提案中概述了旨在详细描述脂质锚定杆菌胺生物合成在模型膜中的生物学机制的方法。该途径中每个酶的全长或可分离结构域将被异种表达、纯化，并在无细胞系统中表征各自的酶活性。然后，纯化的材料将在模型膜中重构，并使用脂质锚定底物进行生化反应。该检测还将用于确定膜，蛋白质跨膜结构域和膜结合的戊烯基焦磷酸分子在杆菌胺生物合成中的作用。

DESCRIPTION (provided by applicant): Biochemical characterization of a process that occurs at the membrane-cytosol interface is inherently challenging due to the bimodal nature of the reactants and products involved. A representative process potentially amenable to biochemical analysis resides in the biosynthesis of bacillosamine from the gram-negative enteropathogen Campylobacter jejuni. In C.jejuni bacillosamine is the first carbohydrate of a heptameric, prenylpyrophosphate-linked glycan that is assembled in the cytosol, transported across the membrane, and N-linked to nascent proteins in the periplasmic space. The locus that codes for enzymes involved in bacillosamine biosynthesis has been cloned and the heptameric glycan identified. However, the roles of the plasma membrane, transmembrane domains of the enzymes, and the membrane bound bactoprenolpyrophosphate molecule during glycan biosynthesis remains unclear. Outlined in this proposal are approaches designed to detail the biological mechanism of lipid anchored bacillosamine biosynthesis in model membranes. Full-length or separable domain constructs for each enzyme in the pathway will be heterologously expressed, purified, and respective enzymatic activity characterized in cell-free systems. Purified material will then be reconstituted in model membranes, and biochemical reactions characterized using lipid anchored substrate. The assays will also be used to identify the roles of the membrane, protein transmembrane domains, and the membrane bound prenylpyrophosphate molecule in bacillosamine biosynthesis.