Dissecting the mechanism by which glycosyltransferases calalyse mannosyl transfer

The linking of sugars to a variety of different proteins has an important influence on the function of cells. The biological catalysts or enzymes that speed up the reactions in which sugars are linked to other molecules are known as glycosyltransferases. Although these enzymes and are industrially and biologically important, they have not been extensively studied because they are difficult to produce in large amounts. In this project we will take advantage of our ability to produce significant quantities of glycosyltransferases that catalyse the transfer of the sugar mannose onto other molecules. The three dimensional structure of these enzymes will be determined and the information will be used to synthesise inhibitors of these biological catalysts and to use molecular engineering techniques to manipulate their biological properties.

Although mannose-containing polymers are widespread in nature, there is a paucity of structural and mechanistic information on the enzymes that catalyze mannosyltransfer. Recent studies by our three groups [Flint et al. (2005) Nat. Struct. Mol. Biol. 12, 608-14] have begun to unravel the structural basis for the catalytic activity and plasticity of substrate recognition of the retaining GDP-Man transferase, mannosylglycerate synthase, which lays a foundation upon which to dissect mannosyl transfer. This application will build upon our studies on mannosylglycerate synthase and a significant additional body of preliminary data, of both retaining and inverting mannosyltransferases (including one of the key enzymes of glycobiology, dolichyl-phosphate -D-mannose synthase) to dissect the mechanism of action and specificity of mannosyltransferases. This will underpin the engineering of these enzymes to increase their utility as biosynthetic tools, underpin novel therapeutic strategies that target glycan decoration and, and, through the modulation of key enzymes that catalyse mannosyltransfer, provide profound insights into cellular function To date, there are no known selective inhibitors of any retaining glycosyltransferases with sufficient potency to allow modulation of the function of these enzymes in vivo. This void has hampered not only the understanding of the role of mannose decoration in biology, but also the exploitation of mannosyl transfer in drug design. The key goals that will be addressed in this project are: (a) Determine how structure dictates specificity and the mechanism of catalysis of mannosyltransferases (b) Exploitation of such information in the design of new enzyme inhibitors which reflect both structural and mechanistic features (c) Interrogation of the evolution of the mechanisms of mannosyl transfer, and its exploitation in the development of novel biocatalysts.