Structure and function of bacterial glycosyltransferase enzymes

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Cell-surface glycoconjugates participate in crucial cellular recognition processes in all biological systems. Glycosyltransferase (GT) enzymes determine the precise order of sugars and linkages in a glycan structure and represent a crucial enzymatic foundation in glycobiology. GTs also have potential biotechnological applications in emerging glycoengineering technologies, where the goal is to produce "glyco-products" with defined chemical and physical characteristics. Bacterial GTs that use sugar nucleotide donors are important in these efforts due to their vast range of specificities. Most GTs are monofunctional (i.e. they catalyze formation of one glycosidic linkage). However, there is a growing list of "polymerases", which contain one or more catalytic sites that add several or many residues to sequentially extend a growing acceptor and these provide our focus. Polymerase GTs are found in prokaryotes and eukaryotes and some form highly important products such as chitin, cellulose and hyaluronic acid. There are currently 96 recognized GT families based on sequence features and (known/predicted) 3-dimensional catalytic-domain folds but there are pivotal gaps in our understanding of features that dictate acceptor/donor specificity. This is a particularly issue with polymerases. A variety of strategies have evolved in bacteria to constrain polymerase activities to favour products of particular lengths, fit for their physiological functions. Once again, the underlying mechanisms are generally poorly understood. Our long-term goal is to understand the relationships between structure and function in polymerase GTs. We seek to establish the architectural principles underlying assembly of efficient multienzyme systems, and elucidate the processes involved in chain-length regulation. These are important fundamental questions in enzymology and microbial cell biology, as well as critical considerations in glycoengineering. Four influential prototype systems have been selected to probe the mechanistic and organizational diversity in bacterial polymerase GTs and each offers unique mechanistic attributes and opportunities for original insight. Some build on an extensive knowledge foundation established in the previous 5-year term, while others are just being introduced. Our experimental approach is to elucidate catalytic activities using in vitro assays with purified proteins (or functional isolated domains from multidomain proteins) and synthetic acceptors. Multienzyme complexes will be isolated and characterized and the critical cellular protein:protein interactions will be mapped. Finally, the proteins and complexes provide targets for structural biology initiatives. The planned multidisciplinary studies are designed to position my group as a global leader in this important area of glycobiology and train the next generation of glycobiologists.**