Structural and functional characterization of phosphoglycosyl transferases from human pathogens

人类病原体磷酸糖基转移酶的结构和功能表征

• 批准号: 10001977
• 负责人: Gregory J Dodge
• 金额: $ 6.53万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10001977
• 关键词: Active Sites; Address; Age; Anabolism; Antibiotic Resistance; Antibiotics; Area; Biochemical; Biological; Biological Assay; Biological Process; Campylobacter; Campylobacter jejuni; Cell Wall; Cell surface; Cocrystallography; Crystallization; Data; Detergents; Development; Diffusion; Environment; Enzymes; Escherichia coli; Exhibits; Family member; Fucose; Future; Glycoconjugates; Glycoproteins; In Vitro; Integral Membrane Protein; Left; Libraries; Link; Lipid Bilayers; Lipids; Lipopolysaccharides; Membrane; Membrane Proteins; Methods; Molecular; Molecular Conformation; Nucleosides; Oryctolagus cuniculus; Pathway interactions; Phase; Phospholipids; Phosphorylcholine; Play; Polymers; Polysaccharides; Proteins; Reporting; Specificity; Staphylococcus aureus; Structure; Structure-Activity Relationship; Styrenes; Substrate Interaction; Substrate Specificity; Sugar Phosphates; Techniques; Technology; Teichoic Acids; Transferase; Uridine Diphosphate Sugars; Virulence; X-Ray Crystallography; antimicrobial; base; colanic acid; copolymer; data warehouse; design; enzyme substrate complex; experimental study; extracellular; human pathogen; immunogenicity; in vivo; inhibitor/antagonist; inorganic phosphate; insight; lipooligosaccharide; luminescence; macromolecule; maleic acid; nanoparticle; novel; pathogen; pathogenic bacteria; prevent; protein purification; protein structure; screening; sugar; vapor; vector

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.

抽象的： 该项目旨在扩大对初始膜相关步骤的机械理解 细菌糖复合物的生物合成。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活力和毒力所必需的，并且是 抗菌设计的有吸引力的目标。例子包括荚膜多糖（CPS）、细胞壁磷壁酸 酸、脂多糖 (LPS) 以及 N- 和 O- 连接糖蛋白。 糖复合物生物合成的第一个膜结合步骤由磷酸糖基转移酶催化 （PGT）。此类酶将糖从 NDP-糖转移到十一碳二烯基磷酸酯脂质上 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。来自 Conpylobacter concisus 的 PGT、PglC 的第一个结构最近被解析，但已结晶 处于活性位点开放且未配体的构象中。 苯乙烯马来酸共聚物（SMALP）和传统洗涤剂都将用于溶解和净化 不同底物选择性的 PGT。化学酶合成将用于生成UDP-糖 PGT 的底物，例如 UDP-diNAcBac 和 UDP-岩藻糖。快速的、基于发光的测定将是 用于表征溶解的靶标和合成底物。脂质立方相 (LCP) 方法将 促进从未离开脂质双层的 SMALP 溶解靶标的结晶。合成基材 将用于浸泡或共结晶实验。这些实验将拓宽我们的理解 PGT 结构-功能关系。数据还将为抑制剂的开发工作提供信息，因为 PGT 仍然是一种 抗菌剂和抗毒剂开发尚未开发的领域。