Structural and functional characterization of phosphoglycosyl transferases from human pathogens

摘要： 该项目旨在扩大对膜相关的初始步骤的机械理解。 细菌糖共轭生物合成。膜蛋白结构在 蛋白质数据库(PDB)，并且仍然是纯化、表征和机制分析的困难目标。 这些生物合成途径的产物是细菌生存和毒力所必需的，并且是 抗菌药物设计具有吸引力的目标。例子包括胶囊多糖(CPS)、细胞壁磷脂 酸、脂多糖以及N-和O-连接的糖蛋白。 糖结合生物合成的第一步是由磷酸糖基转移酶催化的。 (PGTS)。这类酶将糖从NDP-糖转移到十一碳烯基磷脂上 抛锚。不同的PGT表现出不同的底物选择性，其分子决定因素仍然存在 未知。简约弯曲菌PGT的第一个结构PglC最近被解析，但被结晶 在活性中心开放且未连接的构象中。 将使用苯乙烯-马来酸共聚物(SMALP)和传统洗涤剂来增溶和提纯 不同底物选择性的PGTS。化学酶合成法将用于生产UDP-糖 PGTS的底物如UDP-diNAcBac和UDP-岩藻糖。一种基于发光的快速检测将是 用于表征增溶靶标和合成底物。脂质立方相(LCP)方法将 促进从未离开脂质双层的SMALP增溶靶标的结晶。合成底物 将用于浸泡或共结晶实验。这些实验将拓宽我们的理解 前列环素结构与功能的关系。数据还将为开发抑制剂的努力提供信息，因为PGTS仍然是一种 未被开发的抗微生物和抗病毒剂开发领域。

Abstract: This project aims to expand the mechanistic understanding of the initial membrane-associated steps of bacterial glycoconjugate biosynthesis. Membrane protein structures are critically underrepresented in the protein data bank (PDB), and remain difficult targets for purification, characterization and mechanistic analysis. The products of these biosynthetic pathways are required for both bacterial viability and virulence and are attractive targets for antimicrobial design. Examples include capsular polysaccharide (CPS), cell wall teichoic acid, lipopolysaccharide (LPS) and N- and O-linked glycoproteins. The first membrane-bound step of glycoconjugate biosynthesis is catalyzed by phosphoglycosyl transferases (PGTs). This class of enzymes transfers a sugar from an NDP-sugar onto an undecaprenyl phosphate lipid anchor. Different PGTs exhibit different substrate selectivity, the molecular determinants of which remain unknown. The first structure of a PGT, PglC from Campylobacter concisus was solved recently, but crystallized in a conformation in which the active site is open and unliganded. Both Styrene maleic acid copolymer (SMALP) and traditional detergents will be used to solubilize and purify PGTs of differing substrate selectivity. Chemoenzymatic synthesis will be used to generate UDP-sugar substrates for PGTs such as UDP-diNAcBac and UDP-fucose. A rapid, luminescence-based assay will be used to characterize solubilized targets and synthesized substrates. Lipid cubic phase (LCP) methods will facilitate crystallization of SMALP solubilized targets that have never left a lipid bilayer. Synthetic substrates will be utilized for soaking or cocrystallization experiments. These experiments will broaden our understanding of PGT structure-function relationships. Data will also inform efforts to develop inhibitors, as PGTs remain an underexplored area for the development of antimicrobial and antivirulence agents.