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.

抗生素抗性是一个日益增长的世界问题，因此迫切需要开发替代手段 禁用此类病原体。一个目标是细菌糖缀合物的生物合成途径，一种多种类别 大分子在具有挑战性的环境中在细胞壁稳定性中起关键作用的大分子 细菌病原体宿主相互作用。有关涉及En Bloc的酶的结构信息 缺乏糖缀合物的构造，这限制了基于机制的抑制剂设计。这个建议 专注于确定N连接糖基化途径的结构功​​能关系，因为 不同致病性弯曲杆菌细菌之间途径的高度保守性。这 糖基转移酶PGLI通过将分支聚糖附着在聚糖合成的最后一步 未依赖磷酸盐连接的糖聚合物底物。糖的分支位置变化很大 在不同种类的弯曲杆菌和PGLI的作用方式中，尚不清楚。 PGLI有一个 注释的N端GT-A折叠结构域和未知功能的C末端结构域。附加域 可以通过塑造受体糖的活性部位来控制分支聚糖的位置，从而发挥作用 结合和二价阳离子结合以及通过介导蛋白质 - 蛋白质或膜 - 缔合的相互作用。目标1 将通过结构表征来确定PGLI酶中选择性聚糖转移的结构基础 来自几种不同弯曲杆​​菌的PGLI。 AIM 2将专注于确定的催化机制 通过动力学表征和诱变研究，PGLI酶的选择性聚糖转移。 AIM 3将集中精力 通过结构和功能分析发现新型多域聚糖生物合成酶 GT-A折叠超家族。这个目标将通过组合探索GT-A折叠酶的结构空间 具有底物筛选和结构表征的生物信息学。 PGLI的结构表征 将导致对不同弯曲杆​​菌分支聚糖附着的机械基础的理解 物种，使基于结构的抑制剂设计和最终新的抗生素。这项工作的结果将 提高对组织细菌多蛋白复合物的分子机制的理解 糖缀合物生物合成，并允许使用新颖的方法来识别破坏这些化学剂 病原体。