Enzymatic Mechanism of Polysaccharide Length Control by GlfT2

项目摘要/摘要 胞外多糖在生命的各个领域都扮演着重要的角色。细菌多糖是一种 具有多种生物功能的不同类别的大分子，包括与 外部环境和保持细胞壁的完整性。细菌糖基转移酶负责 多糖通过它们在底物专一性和连锁产生上的差异来实现多样性。 多糖的生物合成和伸长可以通过多种机制发生；最不了解的是 过程聚合。加工性是由酶保持受体的能力引起的 无数的延伸步骤。这一过程减少了短链多糖的生产，这可能会 对细菌健康有害。过程性可能代表了一种常见且关键的机制 多糖的生物合成和长度控制。 半乳呋喃糖基转移酶2(GlfT2)产生分枝杆菌半乳糖聚糖的过程被证明是通过 过程机制。分枝杆菌半乳聚糖。是一种基本的结构多糖，其功能是 人类病原体细胞壁结构的组成部分，包括结核分枝杆菌和 麻风分枝杆菌截短半乳糖会降低细胞的适合性，促进周质变薄，并增加 抗生素敏感性。因此，GlfT2的酶处理能力可能确保半乳聚糖有足够的 长度。拟议的研究试图定义GlfT2过程的机制和生物物理 决定产品长度分布的参数。该项目包括酶生产方面的培训。 以及表征、酶动力学分析和酶结构测定。基斯林集团， 化学糖生物学的领导者和麻省理工学院化学系提供了一个丰富的环境 掌握这些研究技能。研究环境也提供了从事科学研究的机会 沟通、文献分析和职业发展。建议的调查结果如下 有望为在其他疾病中发现的进行性糖基转移酶的机制分析提供一个框架 细菌和跨越生命的不同领域。对这类未被充分表征的酶的新见解 这将为抗击现代出现的抗药性细菌提供新的靶点。

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.