Enzymatic Mechanism of Polysaccharide Length Control by GlfT2

GlfT2 控制多糖长度的酶促机制

• 批准号: 10689066
• 负责人: Alan Wylde Carter
• 金额: $ 4.77万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-18 至 2025-08-17
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10689066
• 关键词: Achievement; Anabolism; Antibiotic susceptibility; Bacteria; Bacterial Antibiotic Resistance; Bacterial Polysaccharides; Binding; Biological Assay; Biological Process; Catalysis; Cell Wall; Cells; Cellular Structures; Chemicals; Chemistry; Communication; Disease; Dissociation; Ensure; Environment; Enzyme Kinetics; Enzymes; Exhibits; Future; Galactans; Genus Mycobacterium; Glycobiology; Goals; Growth; Hydrophobicity; In Vitro; Investigation; Kinetics; Lead; Length; Life; Literature; Maps; Measures; Mediating; Modernization; Molecular; Monosaccharides; Mycobacterium leprae; Mycobacterium tuberculosis; Nature; Organism; Orthologous Gene; Physiology; Play; Polymers; Polysaccharides; Process; Production; Proteins; Research; Role; Science; Slide; Specificity; Structure; Substrate Interaction; Substrate Specificity; Testing; Thinness; Training; Variant; Work; X-Ray Crystallography; antibiotic design; arabinogalactan; bacterial fitness; biophysical properties; career development; combat; enzyme structure; extracellular; fitness; glycosyltransferase; human pathogen; in vitro activity; insight; macromolecule; mycobacterial; novel; periplasm; polymerization; preservation; simulation; skills; sugar

PROJECT SUMMARY/ABSTRACT Extracellular polysaccharides play critical roles across all domains of life. Bacterial polysaccharides are a diverse class of macromolecules with multiple biological functions, including mediating interactions with the external environment and preserving cell wall integrity. Bacterial glycosyltransferases are responsible for polysaccharide diversity through their differences in substrate specificity and linkage production. Polysaccharide biosynthesis and elongation can occur by multiple mechanisms; the least understood is processive polymerization. Processivity is elicited from an enzyme’s ability to retain the acceptor through numerous elongation steps. This process reduces the production of short-length polysaccharides, which could be harmful to bacterial fitness. Processivity may represent a common and critical mechanism for polysaccharide biosynthesis and length control. Production of the mycobacterial galactan by galactofuranosyltransferase 2 (GlfT2) was shown to proceed by a processive mechanism. The galactan of Mycobacterium spp. is an essential structural glycan, functioning as a component of the cell wall structure of human pathogens including Mycobacterium tuberculosis and Mycobacterium leprae. Galactan truncation decreases cell fitness, promotes periplasm thinning, and increases antibiotic susceptibility. Therefore, enzymatic processivity by GlfT2 likely ensures the galactan is of sufficient length. The proposed studies seek to define the mechanism of GlfT2 processivity and the biophysical parameters that dictate product length distributions. This project encompasses training in enzyme production and characterization, enzyme kinetics assays, and enzyme structure determination. The Kiessling group, leaders in chemical glycobiology, and the Department of Chemistry at MIT provide a rich environment to acquire these research skills. The research environment also offers opportunities to engage in science communication, literature analysis, and career development. The results from the investigations proposed are expected to provide a framework for mechanistic analysis of processive glycosyltransferases found in other bacteria and across the different domains of life. New insights into this under-characterized class of enzymes will provide novel targets to combat the modern emergence of antibiotic-resistant bacteria.

项目总结/摘要 细胞外多糖在生命的各个领域都发挥着关键作用。细菌多糖是一种 具有多种生物学功能的多种大分子，包括介导与 外部环境和保持细胞壁完整性。细菌糖基转移酶负责 多糖多样性通过它们在底物特异性和连接产生方面的差异来实现。 多糖的生物合成和延伸可以通过多种机制发生;最不了解的是 连续聚合持续加工能力是由酶通过以下方式保留受体的能力引起的： 许多延伸步骤。这个过程减少了短长度多糖的产生， 对细菌健康有害。持续性可能代表了一种常见的和关键的机制， 多糖生物合成和长度控制。 通过半乳糖呋喃糖基转移酶2（GlfT 2）生产分枝杆菌半乳聚糖， 进行机制分枝杆菌属的半乳聚糖。是一种重要的结构聚糖， 包括结核分枝杆菌在内的人类病原体的细胞壁结构的组分， 麻风分枝杆菌半乳聚糖截短降低细胞适应性，促进周质变薄， 抗生素敏感性因此，GlfT 2的酶促持续合成能力可能确保半乳聚糖具有足够的 长度所提出的研究试图定义GlfT 2持续合成能力的机制和GlfT 2的生物物理机制。 决定产品长度分布的参数。该项目包括酶生产培训 和表征、酶动力学测定和酶结构测定。Kiessling集团， 化学糖生物学的领导者和麻省理工学院的化学系提供了丰富的环境， 掌握这些研究技能。研究环境也提供了从事科学的机会 沟通，文献分析和职业发展。拟议调查的结果如下： 预期提供一个框架，机制分析的进行性糖基转移酶中发现的其他 细菌和生命的不同领域。对这类特征不足的酶的新见解 将提供新的目标，以打击现代出现的抗药性细菌。