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