Grant: Discovery Grants Program - Individual

Our bioorganic chemistry program has the broad goals of furthering an understanding of the physiological roles played by carbohydrate-manipulating enzymes and using these catalysts to perform novel chemistry. The proposed research will use: synthetic chemistry to access novel substrates; mutagenesis and kinetic analyses to probe and evolve catalyst function; and NMR spectroscopy to provide structural insight into protein-ligand interactions. Having obtained preliminary data on several model enzyme systems in our previous NSERC funding period, we will focus upon developing screening and selection methodologies for two families of enzymes with an intent to identify novel chemical reactivity. Two projects are described more fully whilst two are described in lesser detail due to space limitations. 1. We will develop a new selection strategy for evolving glycosynthases (EryBI D273G) that form beta-(1,2)- glycosidic linkages. The unique feature of this project is the recognition that improved glycosynthases will inactivate macrolide antibiotics through glycosylation and ensure growth of their host cell. This will enable us to select for different donor and acceptor specificities. 2. We will characterize in vitro a 2,6-dideoxyglycosyltransferase with a demonstrated broad substrate specificity and manipulate the glycosylation mechanism using directed evolution to convert the enzyme from an O-glycosyltransferase into a C-glycosyltransferase. 3. We will engineer substrate for nucleotidylyltransferases in order to provide access to bifunctional glycosyltransferase probes with bioorthogonal reactivity as cellular tags or as glycosyltransferase inhibitors. 4. We will chemoenzymatically synthesize analogues of UDP-GlcNAc, as potential mechanism-based inhibitors of cell wall biosynthetic enzymes, exploiting our observations in the substrate specificity of nucleotidylyltransferases.