UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Mechanisms and engineering of glycosyltransferases Lairson, Luke Lee

Abstract

In order to gain insight into the natural evolution of enzyme mechanism and to increase the utility of a class of sugar modifying enzymes known as glycosyltransferases, several representative enzymes were subjected to mechanistic and engineering studies. The chemical and kinetic mechanisms of the GT-A fold inverting sialyltransferase Cst II were investigated by detailed kinetic analysis, protein X-ray crystallography and mutagenesis. This enzyme catalyzes the general SN[subscript]2-like direct displacement mechanism used by all inverting glycosyltransferases. However, the chemical strategies utilized to facilitate reaction are more akin to those of the GT-B fold enzymes, indicating a convergence in mechanism between these two clans of enzymes. By analogy to retaining glycosidases, retaining glycosyltransferases had been thought to use a double displacement mechanism involving an enzymatic nucleophile. However, a comparison of the X-ray crystal structures of multiple retaining glycosyltransferases indicates a complete lack of conserved structural architecture in the region that would be occupied by this critical catalytic residue. This lack of conserved architecture, precedence for cationic enzymatic mechanisms, and the inherent differences in reactivities of glycosyltransferase and glycosidase substrates all support a notion that the majority of retaining glycosyltransferases utilize a mechanism involving the formation of a short-lived ion pair intermediate species. However, a protein engineering approach was used to explore the possibility of nucleophilic catalysis in the retaining galactosyltransferase LgtC. The results of this work led to the first direct observation of a catalytically relevant covalent glycosyl-enzyme intermediate for a retaining glycosyltransferase. It was demonstrated that catalytically active LgtC could be displayed on the surface of M13 bacteriophage as a pIII fusion protein. Further, LgtC phage display was successfully performed in the context of a water-in-oil emulsification procedure that may have significant utility in the development of directed evolution screening approaches. A substrate engineering strategy was developed which allowed the substrate specificity of wild type LgtC to be broadened to allow exclusive formation of α-1,2, α-1,3 or α-1,4 linkages at synthetically useful rates with various alternative acceptor substrates. Finally, it was demonstrated that Cst II and LgtC will utilize alternative donor substrates in the presence of their respective natural nucleotide products.

Item Media

Item Citations and Data

License

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

Usage Statistics