Characterization of bacillosamine synthesis in C. jejuni

描述（由申请人提供）：由于所涉及的反应物和产物的双峰性质，在膜-胞质溶胶界面发生的过程的生物化学表征具有固有的挑战性。一个可能适合于生化分析的代表性过程在于从革兰氏阴性肠道病原体空肠弯曲杆菌生物合成杆菌胺。在空肠弯曲杆菌中，杆菌胺是七聚体焦磷酸异戊烯基连接聚糖的第一个碳水化合物，其在胞质溶胶中组装，跨膜转运，并与周质空间中的新生蛋白质N-连接。已克隆了编码参与杆菌胺生物合成的酶的基因座，并鉴定了七聚体聚糖。然而，在聚糖生物合成过程中质膜、酶的跨膜结构域和膜结合的bactoprenolpyrophosphate分子的作用仍不清楚。在这个建议中概述的方法，旨在详细的生物机制的脂质锚定的杆菌胺生物合成的模型膜。途径中每种酶的全长或可分离结构域构建体将异源表达、纯化，并且在无细胞系统中表征各自的酶活性。然后将纯化的材料在模型膜中重构，并使用脂质锚定底物表征生化反应。该测定还将用于鉴定膜、蛋白质跨膜结构域和膜结合的异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.