| Among the many biomolecules found in nature, such as amino acids and DNA, carbohydrates are among the most important and abundant. The assembly of carbohydrates to build various oligosaccharides or glycans is of considerable interest in the glycobiology community, as understanding, controlling, and manipulating such assemblies can yield many interesting insights. This is because glycans are directly involved with many critical biological processes such as molecular recognition, cell-to-cell communication, protein folding and stability, as well as being involved many unique disease state phenotypes. The assembly of these glycans is commonly accomplished by a class of enzymes called glycosyltransferases, and it is these enzymes which have been exploited in vitro for the synthesis of attractive oligosaccharides. However, the identification, characterization, and subsequent exploitation of a glycosyltransferase is a very tedious and long process which stems from the root problem that knowing the primary function of a glycosyltransferase must be determined experimentally. This is because glycans are not the result of primary gene products, and identifying the function and specificity of a glycosyltransferase is not straight forward. In this thesis, the work has been focusing on developing a method by which function can be quickly assigned to a putative glycosyltransferase, as well as characterizing several bacterial glycosyltransferases, and lastly exploiting glycosyltransferase functions to perform various functions;Chapter 1 provides a general introduction to the function of glycans, the function and biosynthesis of cell surface oligosaccharides, an overview of the methodologies used to determine glycosyltransferase functions, and lastly a review of chemical approaches to study and exploit oligosaccharide biosynthesis.;Chapter 2 and 3 are primarily associated with characterization of bacterial glycosyltransferases and biosynthesis of O-antigen structures in two different strains of E. coli. Chapter 2 focuses on investigating the biosynthesis of the O-antigen repeating unit in E. coli O127 and E. coli O128; whereas Chapter 3 focuses on the detailed biochemical characterization of WbnI, a bacterial homologue of the important human blood group B galactosyltransferase.;In Chapter 4, a high throughput technology was developed by which a putative glycosyltransferase can be quickly cloned, expressed, and relative substrate specificity determined. This work utilizes ligation independent cloning, in vitro protein expression, and SAMDI mass spectrometry along with a library of donor/acceptor substrates, by which enzymatic activity of putative glycosyltransferases is assessed.;In Chapter 5, glycosyltransferases and sugar nucleotide biosynthesis are exploited both in vivo and in vitro for the synthesis of interesting and novel oligosaccharides.;Finally, Chapter 6 summarizes the main results of all the studies included in this thesis, and also provides further directions for each project. |
