Defining structure and function of GT-A fold enzymes in bacterial glycan assembly

定义细菌聚糖组装中 GT-A 折叠酶的结构和功能

• 批准号: 10752020
• 负责人: Hayley Knox
• 金额: $ 6.91万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752020
• 关键词: Acceleration; Active Sites; Anabolism; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Antibiotic Resistance; Binding; Binding Sites; Biochemical; Bioinformatics; Biological Assay; C-terminal; Campylobacter; Categories; Cations; Cell Wall; Chemical Agents; Chimeric Proteins; Classification; Complex; Cryoelectron Microscopy; Deltaproteobacteria; Development; Divalent Cations; Environment; Enzyme Interaction; Enzyme Kinetics; Enzymes; Epsilonproteobacteria; Family; Glycoconjugates; Kinetics; Knowledge; Life; Link; Lipids; Location; Maps; Mediating; Membrane; Metals; Methodology; Molecular; Multiprotein Complexes; Mutagenesis; N-terminal; Orthologous Gene; Outcome; Pathogenicity; Pathway interactions; Play; Polysaccharides; Positioning Attribute; Protein Glycosylation; Proteins; Reaction; Resistance; Roentgen Rays; Role; Scanning; Screening Result; Shapes; Specificity; Structure; Structure-Activity Relationship; Styrenes; Therapeutic; Virulence; Work; X-Ray Crystallography; design; dimer; enzyme activity; experimental study; global health; glycosylation; glycosyltransferase; inhibitor; inorganic phosphate; link protein; macromolecule; maleic acid; nano; novel; novel strategies; novel therapeutics; particle; pathogen; pathogenic bacteria; protein complex; screening; sugar

Antibiotic resistance is a growing world problem and thus there is an urgent need to develop alternative means to disable such pathogens. One target is the biosynthetic pathway of bacterial glycoconjugates, a diverse class of macromolecules that play pivotal roles in cell-wall stability in challenging environments and in mediating bacterial pathogen-host interactions. Structural information about the enzymes involved in the en bloc construction of the glycoconjugates is lacking, which limits mechanism-based inhibitor design. This proposal focuses on determining the structure-function relationships of the N-linked glycosylation pathway, because of the high conservation of the pathway amongst the different pathogenic Campylobacter bacterium. The glycosyltransferase PglI catalyzes the final step in glycan synthesis through attachment of a branching glycan to the undecaprenyl phosphate-linked glycopolymer substrate. The branching position of the sugar varies widely amongst the different species of Campylobacter and the mode of action of PglI is not known. PglI has an annotated N-terminal GT-A fold domain and a C-terminal domain of unknown function. The additional domain may play a role in controlling the location of the branching glycan by shaping the active site for acceptor sugar binding and divalent cation binding and by mediating protein-protein or membrane-associate interactions. Aim 1 will identify the structural basis of selective glycan transfer in PglI enzymes through structural characterization of PglI from several different Campylobacter species. Aim 2 will focus on determining the catalytic mechanism of selective glycan transfer of PglI enzymes by kinetic characterization and mutagenesis studies. Aim 3 will focus on the discovery of novel multidomain glycan biosynthetic enzymes through structural and functional profiling of the GT-A fold superfamily. This aim will explore the structural space of GT-A fold enzymes by combining bioinformatics with substrate screening and structural characterization. The structural characterization of PglI will lead to an understanding of the mechanistic basis for branching glycan attachment in different Campylobacter species, enabling structure-based design of inhibitors and ultimately new antibiotics. The results of this work will advance the understanding of the molecular mechanisms that organize the multiprotein complexes of bacterial glycoconjugate biosynthesis and allow for novel approaches for identifying chemical agents that disrupt these pathogens.

抗生素耐药性是一个日益严重的世界性问题，因此迫切需要开发替代方法。 使这些病原体失效。一个目标是细菌糖结合物的生物合成途径，这是一种不同的类别 在具有挑战性的环境中对细胞壁的稳定性起关键作用的大分子 细菌病原体与宿主的相互作用。关于参与整个区块的酶的结构信息 缺乏糖共轭化合物的构建，这限制了基于机制的抑制剂设计。这项建议 重点是确定N-连接糖基化途径的结构-功能关系，因为 不同致病弯曲菌之间途径的高度保守性。这个 糖基转移酶PglI通过将支链糖链连接到糖链上，催化合成糖链的最后一步 该十一碳烯基磷酸连接的糖共聚物底物。糖的分枝位置变化很大 在不同种类的弯曲杆菌中，PglI的作用方式尚不清楚。Pgli有一个 注释的N-末端GT-A折叠域和未知功能的C-末端结构域。附加域 可能通过塑造受体糖的活性部位来控制分枝多糖的位置 结合和二价阳离子结合以及通过调节蛋白质-蛋白质或膜-缔合物的相互作用。目标1 将通过对PglI酶的结构特征的表征来确定PglI酶中选择性糖链转移的结构基础 PglI来自几个不同的弯曲菌物种。目标2将集中于确定其催化机理 通过动力学表征和诱变研究PglI酶的选择性糖链转移。目标3将专注于 通过结构和功能图谱发现新的多结构域多糖生物合成酶 GT-A Fold超家族。这一目标将探索GT-A折叠酶的结构空间 生物信息学与底物筛选和结构表征。PglI的结构表征 将有助于理解不同弯曲杆菌中支化多聚糖附着的机制基础 新品种，使基于结构的抑制剂设计成为可能，并最终产生新的抗生素。这项工作的结果将是 促进对细菌多蛋白复合体的分子机制的理解 糖共轭生物合成，并允许采用新的方法来识别破坏这些 病原体。