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.
