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，发生在膜-细胞液界面的过程的生化表征本身就具有挑战性。一个可能适合生化分析的代表性过程存在于从革兰氏阴性肠病原菌空肠弯曲杆菌生物合成杆菌三胺。在空肠弯曲菌中，杆菌胺是七聚体的第一个碳水化合物，它是戊基焦磷酸连接的多糖，在胞浆中组装，跨膜运输，并与周质空间中的新生蛋白质连接。参与杆菌胺生物合成的酶的编码基因已经被克隆，并鉴定了七聚体多糖。然而，质膜、酶的跨膜结构域以及膜结合的焦磷酸杆菌分子在糖链生物合成中的作用尚不清楚。本提案概述了旨在详细说明模型膜中脂质锚定杆菌胺生物合成的生物学机制的方法。途径中每个酶的全长或可分离结构域将被异源表达、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组，并使用脂类锚定底物表征生化反应。这些分析也将被用来确定膜、蛋白质跨膜结构域和膜结合的戊二烯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.