Structure/Function Analysis of Trans-Sialidase

唾液酸是多种生物体和组织中糖蛋白和糖脂上的必需末端糖，糖及其凝集素在正常蛋白质、细胞和细胞基质相互作用中起作用。最近已经描述了一种新型的唾液酸代谢酶，其非常有效地将唾液酸从一种碳水化合物供体底物移动到另一种碳水化合物受体。这种酶，转唾液酸酶，在转移反应中不使用核苷酸糖底物，如高尔基体的唾液酸转移酶，而是切割糖蛋白和糖脂上发现的末端β 2，3连接的唾液酸残基，并将糖转移到合适的受体。这种转移反应的机制的性质是未知的。还描述了来自相关生物体的另一种酶，其能够仅作为唾液酸酶作用于相同的供体底物，即，它将裂解唾液酸但不将其转移到另一种碳水化合物受体。本研究比较了转唾液酸酶和唾液酸酶蛋白质的氨基酸序列与已知二级和三级结构的细菌和病毒唾液酸酶。采用分子生物学方法，通过交换两种基因类型的亚区，并通过使用位点特异性诱变，在每种情况下随后测定新蛋白质产物的酶活性，来确定酶的哪些区域导致酶功能的差异。还将创建重组构建体以过表达这两种酶，用于旨在通过蛋白质晶体分析推断酶的三级结构的协作工作。 转唾液酸酶和一般唾液酸酶的序列和结构域结构的知识，然后将被应用于尝试创建具有新的底物特异性的唾液酸转移酶。这种新的酶将具有广泛的科学和商业潜力，用于酶促合成目前必须有机合成的复杂碳水化合物材料。 %%% 糖（或碳水化合物）分子对各种生物系统的功能至关重要。 细胞表面最常见的糖类型之一是唾液酸。 许多不同的正常发育和免疫细胞-细胞相互作用需要唾液酸;许多不同的酶添加或去除唾液酸。最近描述了一种来自原生动物生物体的新型酶，其有效地将唾液酸转移到受体分子。这种酶，转唾液酸酶，首先去除唾液酸，然后将其转移到合适的碳水化合物受体分子。来自相关生物的另一种酶仅进行转唾液酸酶反应的第一步（唾液酸酶），并且这两种酶的结构的比较应导致关于转唾液酸酶的独特唾液酸转移反应机制的建议。这项研究涉及到原生动物的转唾液酸酶和唾液酸酶的特性和比较。有了这些信息，应该可以修饰其他唾液酸酶来进行转移反应，并产生将这些糖转移到受体的其他酶。这种酶可用于大规模反应以产生新的含碳水化合物的材料。

Sialic acid is an essential terminal sugar on glycoproteins and glycolipids in a variety of organisms and tissues, with roles for the sugar and its lectins in normal protein protein, cell cell and cell matrix interactions. A novel type of sialic acid metabolizing enzyme has recently been described that very efficiently moves sialic acid from one carbohydrate donor substrate to another carbohydrate acceptor. This enzyme, trans sialidase, does not use nucleotide sugar substrates in the transfer reaction, as do sialyltransferases of the Golgi apparatus, but instead it cleaves terminal (2,3 linked sialic acid residues found on glycoproteins and glycolipids, and transfers the sugar to suitable acce ptors. The nature of the mechanism of this transfer reaction is unknown. Another enzyme has also been described from a related organism which is capable of acting only as a sialidase on the same donor substrates, i.e., it will cleave sialic acid but not transfer it to another carbohydrate acceptor. This research compares the amino acid sequences of the trans sialidase and sialidase proteins to bacterial and viral sialidases of known secondary and tertiary structure. A molecular biological approach is taken to determine which regions of the enzymes account for the differences in enzymatic function by reciprocally exchanging subregions of the two gene types, and by using site specific mutagenesis, in each case followed by assays of the novel protein products for enzymatic activities. Recombinant constructs will also be created to overexpress the two enzymes for collaborative work aimed at deducing the enzymes' tertiary structures via protein crystal analysis. Knowledge of the sequence and domain structure of trans sialidase, and sialidases in general, will then be applied in attempts to create sialic acid transferring enzymes with novel substrate specificities. Such novel enzymes would have extensive scientific and commercial potential for the enzymatic synthesis of complex carbohydrate materials, which at the present time must be organically synthesized. %%% Sugar (or carbohydrate) molecules are critical to the functioning of a variety of biological systems. One of the most common types of sugars on the cell surface is sialic acid. Many different normal developmental and immunological cell-cell interactions require sialic acid; and a number of different enzymes add or remove sialic acid. A novel enzyme, from a protozoan organism, has recently been described that efficiently transfers sialic acid to an acceptor molecule. This enzyme, trans sialidase, first removes sialic acid, and then transfers it to suitable carbohydrate acceptor molecules. Another enzyme from a related organism carries out on ly the first (sialidase) step of the trans sialidase reaction, and a comparison of the structures of these two enzymes should lead to suggestions about the unique sialic acid transfer reaction mechanism of trans sialidase. This research involves characterizing and comparing the protozoan trans sialidase and sialidase enzymes. With this information it should be possible to modify other sialidases to carry out the transfer reaction, and to create other enzymes that transfer these sugars to acceptors. Such enzymes could be used in large scale reactions to create new carbohydrate containing materials.