Grant: Discovery Grants Program - Individual

The structures, synthesis, and functions of glycosylated macromolecules (glycoconjugates) underpin the rapidly evolving multidisciplinary field of glycobiology. Cell-surface glycoconjugates play roles in a remarkable range of cellular and molecular recognition events important in cell growth, development, and disease. Variations in monosaccharide components, anomeric configurations, and linkage positions afford far greater diversity in glycans than is possible with proteins, in order to guide specific molecular interactions. While eukaryotes use a small subset of nature's monosaccharides, bacteria use many more building blocks in an impressive catalogue of glycan structures, yet the overall assembly strategies share some basic principles. Synthesis of glycan structures is performed by glycosyltransferase (GT) enzymes with precise specificities, representing the enzymatic foundation of glycobiology. Currently, most well-studied GTs are monofunctional enzymes (i.e. a single active site catalyzes the formation of a single precise glycosidic bond). However, in bacteria, we are discovering increasing examples of polymerizing GTs (polymerases) with multiple catalytic domains, that are sufficient for chain extension of glycans with varying structural complexity. Some of these enzymes are paired with cognate chain-terminating enzymes, whose activity is controlled by a molecular ruler to regulate the distribution of chain lengths in the glycan products. Despite the importance of GTs in biology, and their increasing utility in emerging glycoengineering technologies, there are still significant gaps in our knowledge concerning their mechanisms and structural features that dictate their specificity. The need for this fundamental insight provides the rationale and focus for this proposal. In the next five years, we will use innovative interdisciplinary approaches to address critical questions concerning GT polymerases on a scale of enquiry from molecules to cells. We propose to: (i) define new enzyme structures and reaction chemistries with broad impact in microbial glycobiology; (ii) provide the first detailed insight into the architectural principles of prototype enzyme complexes; and (iii) establish the spatial distribution of these complexes in bacterial cells. Our plans feature broadly representative prototype enzymes/complexes, where we have established the essential foundational understanding; each is selected to address an original question. Our approach is multidisciplinary and collaborative, offering an ideal environment for training all levels of HQP. It leverages our wealth of established capabilities in microbial cell biology, glycan structure, and biochemistry, and is supported by existing productive collaborations with national and international leaders in carbohydrate chemistry and structural biology. These studies are positioned to reinforce our leadership in the international glycobiology research community.