Structure/Function Analysis of Trans-Sialidase

转唾液酸酶的结构/功能分析

• 批准号: 9418190
• 负责人: Daniel Eichinger
• 金额: $ 27万
• 依托单位: New York University Medical Center
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9418190&HistoricalAwards=false
• 关键词: Structure; Function; Analysis; Trans; Sialidase

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.

唾液酸是多种生物和组织中糖蛋白和糖脂上的一种必需的末端糖，在正常蛋白质、细胞和细胞基质的相互作用中发挥作用。最近描述了一种新型的唾液酸代谢酶，它能非常有效地将唾液酸从一个碳水化合物供体底物转移到另一个碳水化合物受体。这种酶，反式唾液酸酶，不像高尔基体的唾液酸基转移酶那样，在转移反应中使用核苷酸糖底物，而是裂解位于糖蛋白和糖脂上的末端(2，3连接的唾液酸残基)，并将糖转移到合适的受体。这种转移反应的机理的性质尚不清楚。还描述了另一种来自相关生物体的酶，它只能在相同的供体底物上作为唾液酸酶起作用，即它将裂解唾液酸，但不能将其转移到另一个碳水化合物受体。这项研究将反式唾液酸酶和唾液酸酶蛋白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进行了比较。采用分子生物学的方法，通过相互交换两种基因类型的亚区，并使用定点突变，然后对新的蛋白质产物进行酶活性分析，来确定酶的哪些区域解释了酶功能的差异。还将创建重组构建物来过表达这两种酶，用于协作工作，旨在通过蛋白质晶体分析推断酶的三级结构。反式唾液酸酶的序列和结构的知识，以及唾液酸酶的一般知识，然后将被应用于尝试创造具有新的底物特异性的唾液酸转移酶。这种新型的酶对于目前必须有机合成的复杂碳水化合物材料的酶合成具有广泛的科学和商业潜力。糖(或碳水化合物)分子对多种生物系统的功能至关重要。细胞表面最常见的糖类之一是唾液酸。许多不同的正常发育和免疫细胞间的相互作用需要唾液酸；许多不同的酶增加或去除唾液酸。最近描述了一种来自原生动物有机体的新型酶，它可以有效地将唾液酸转移到受体分子上。这种酶，反式唾液酸酶，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。另一种来自相关生物的酶只执行反式唾液酸酶反应的第一步，通过对这两种酶的结构的比较，可以得出反式唾液酸酶独特的唾液酸转移反应机理。这项研究涉及原生动物反式唾液酸酶和唾液酸酶的特性和比较。有了这些信息，应该可以修改其他唾液酸酶来进行转移反应，并产生其他酶来将这些糖转移到受体上。这种酶可以用于大规模反应，以产生新的含碳水化合物的材料。