Structure/Function Analysis of Trans-sialidase

9727826艾辛格转唾液酸酶是由原生动物克氏锥虫产生的一种新型唾液酸代谢酶。这种酶非常有效地将唾液酸从一个碳水化合物供体转移到另一个碳水化合物受体分子。因此，该酶首先作为唾液酸酶发挥作用，从供体中去除α2，3连接的唾液酸，然后通过将唾液酸重新连接到以β-半乳糖终止的受体上而作为唾液酸基转移酶发挥作用。这种独特的两步糖转移反应的机制尚不清楚。研究人员最近确定了该酶的两个结构域，这两个结构域参与了切割和转移反应。与细菌唾液酸酶序列相似的N-末端结构域和与Fn3结构域序列相似的C-末端结构域都有参与酶反应的氨基酸。研究人员现在将通过对编码反式唾液酸酶和相关唾液酸酶的基因进行结构域交换、位点特异性和随机突变来扩展对这两个结构域的分析，以更全面地了解该酶的结构/功能方面。研究人员还将比较各种其他低等真核生物的反式唾液酸酶和唾液酸酶的序列，以探索这些唾液酸代谢酶结构的保守范围。唾液酸是一种重要的糖类，存在于许多生物活性分子中。它通常被添加到蛋白质中，因为它们是由细胞内的酶合成的。最近，一种名为反式唾液酸酶的独特酶被发现，它将这种糖从一个分子转移到另一个分子，使用已经合成的糖蛋白作为底物，并在细胞外发挥作用。这种酶是如何将糖从一个分子转移到另一个分子的，目前尚不清楚。详细了解糖转移反应的步骤以及与糖供体和受体底物相互作用的酶域的结构，将允许设计酶的抑制剂，以确定其在制造糖的生物体中的作用，并将这种酶和其他相关酶直接改变为新型唾液酸代谢酶，这些酶本身已经具有科学价值和商业价值。

9727826 Eichinger Trans-sialidase is a novel type of sialic acid metabolizing enzyme, made by the protozoan Trypanosoma cruzi. this enzyme very efficiently moves sialic acid from one carbohydrate donor to another carbohydrate acceptor molecule. The enzyme thus first functions as a sialidase, by removing alpha 2,3-linked sialic acid from donors, and then as a sialyltransferase, by reattaching the sialic acid to beta-galactose-terminated acceptors. The mechanism of this unique two step sugar transfer reaction is unknown. The investigator has recently identified two domains of the enzyme which are involved in the cleavage and transfer reactions. An N-terminal domain with sequence similarity to bacterial sialidases and a C-terminal domain with weak sequence similarity to a Fn3 domain each have amino acids involved in the enzymatic reactions. The investigator will now extend the analysis of these two domains, using domain-swap, site-specific, and random mutagenesis of the genes encoding trans-sialidase and related sialidases, to gain a more complete understanding of the structure/function aspects of the enzyme. The investigator will also compare the sequences of trans-sialidases and sialidases from a variety of other lower eucaryotes, to explore the range of conservation of structure in these sialic acid metabolizing enzymes. Sialic acid is an important type of sugar found on many biologically active molecules. It is usually added to proteins as they are synthesized by enzymes found inside cells. Recently, a unique enzyme, called trans-sialidase, was found which moves this sugar from one molecule to another, uses already synthesized glycoproteins as substrates, and acts outside of cells. How the enzyme transfers sugars from one molecule to another is not known. A detailed understanding of the steps in the sugar transfer reaction, and the structure of the enzyme domains that interact with the sugar donor and acceptor substrates, would allow for the design of inhibitors of the enzyme with which to define its role in the organisms that make it, and for the directed alteration of this and other related enzymes, themselves already scientifically and commercially valuable, into novel types of sialic acid metabolizing enzymes.