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

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手段 使这些病原体失效。一个目标是细菌糖复合物的生物合成途径， 在挑战性环境中对细胞壁稳定性起关键作用的大分子， 细菌病原体-宿主相互作用有关参与整体酶的结构信息 缺乏糖缀合物的构建，这限制了基于机制的抑制剂设计。这项建议 侧重于确定N-连接糖基化途径的结构-功能关系，因为 在不同的致病性弯曲杆菌属细菌中该途径的高度保守性。的 糖基转移酶PglI通过将分支聚糖连接到 磷酸十一异戊二烯酯连接的糖基共聚物底物。糖的分支位置变化很大 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折叠结构域和未知功能的C-末端结构域。的另外的结构域 通过使受体糖的活性位点成形， 结合和二价阳离子结合以及介导蛋白质-蛋白质或膜-缔合相互作用。要求1 将通过结构表征确定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属物种的PglI。目标2将集中在确定催化机制 通过动力学表征和诱变研究来选择性地转移PglI酶的聚糖。目标3将重点 通过结构和功能分析发现新的多结构域聚糖生物合成酶， GT-A折叠超家族。这一目标将探索结构空间的GT-A折叠酶结合 生物信息学与底物筛选和结构表征。PglI的结构表征 将导致对不同弯曲杆菌中分支聚糖附着的机制基础的理解 这使得基于结构的抑制剂设计成为可能，并最终成为新的抗生素。这项工作的结果将 推进对组织细菌多蛋白复合物的分子机制的理解， 本发明提供了用于糖缀合物生物合成的新方法，并且允许用于鉴定破坏这些生物合成的化学试剂的新方法。 病原体

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.