Structural and functional studies of enzymes involved in glycosaminoglycan synthesis and degradation

本研究旨在研究糖胺聚糖（GAGs）合成和降解过程中酶的结构和功能特征。GAGs是带负电荷的线性多糖，对于提供结缔组织的弹性、生长因子和其他蛋白质的储存、参与组织修复和通过受体发出信号至关重要。gag的存在通常与高等生物有关；然而，它们并不是真核生物独有的。一些特殊的微生物也能产生这些聚合物的非硫酸盐形式。哺乳动物的gag通过水解酶的作用进行快速周转。细菌进化出了使用一种分解机制来降解GAGs的酶，即多糖分解酶。这些酶帮助细菌通过降解表面多糖或利用gag作为食物来源进入哺乳动物细胞。**在此，我们建议扩大对GAG加工酶的研究，并提供透明质酸合成酶、GAG硫转移酶和多糖裂解酶的结构、功能和酶学表征。所有这些蛋白质也可能用于合成治疗疾病的设计寡糖和表征gag的特定工具。**短期目标是：**1。确定PL12和PL17家族多糖裂解酶的结构和催化机制，并建立其与其他PL家族的进化关系。* * 2。通过结构研究和定点诱变建立透明质酸合酶中两个独立的糖基转移酶活性位点协调交替添加葡萄糖醛酸盐和葡萄糖胺的机制。* * 3。目的：确定哺乳动物硫转移酶的管腔片段的结构，并确定其对硫酸盐加成的位置特异性特征

This proposal aims at a structural and functional characterization of enzymes involved in synthesis and degradation of glycosaminoglycans (GAGs). GAGs are negatively charged linear polysaccharides that are essential for providing resilience to connective tissues, storage of growth factors and other proteins, participate in tissue repair and signaling through receptors. The presence of GAGs is usually associated with higher organisms; however, they are not unique to eukaryotes. Several specialized microorganisms also produce unsulfated forms of these polymers. Mammalian GAGs undergo rapid turnover through action of hydrolases. Bacteria evolved enzymes that use a lytic mechanism to degrade GAGs, namely polysaccharide lyases. These enzymes help bacteria to gain access to mammalian cells by degrading the surface polysaccharides or to utilize GAGs as a food source.**Here we propose to expand our research into GAG processing enzymes and provide structural, functional and enzymological characterization of hyaluronan synthase, GAG sulfotransferases and polysaccharide lyases. All these proteins may also potential applications in synthesis of designer oligosaccharides for treatment of diseases and specific tools for characterization of GAGs.**The short-term objectives are:**1. To determine the structures and catalytic mechanisms of polysaccharide lyases from families PL12 and PL17 and establish their evolutionary relationship to other PL families. **2. To establish through structural investigations and site-directed mutagenesis the mechanism of a concerted alternate addition of a glucuronate and a glucosamine by two independent glycosyltransferase active sites within hyaluronan synthase. **3. To determine the structures of lumenal fragments of mammalian sulfotransferases and identify features which define their positional specificity for sulfate addition.**