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.
