Structural and mechanistic studies of specific carbohydrate processing enzymes

Abstract

Carbohydrates are one of the most abundant biomolecules on earth. The functions of carbohydrates are determined by their molecular structure. The structure of carbohydrates is controlled by carbohydrate processing enzymes. Carbohydrate processing enzymes are able to transfer, remove and remodel sugar moieties. The work described in the thesis focuses on an interesting form of protein glycosylation in eukaryotes and the glycosyltransferases responsible for its catalysis, and a family of glycoside hydrolases of biotechnological and medicinal interest.

he attachment of a carbohydrate to a protein is known as protein glycosylation and is catalysed by glycosyltransferases. Proteins are glycosylated through the formation of bonds to three different acceptor atoms: nitrogen, oxygen or carbon. The work presented here describes studies on the glycosyltransferases responsible for C-mannosylation of proteins, in particular their acceptor specificity and the structures of the transferases. Studies were done to attempt expression and purification of the four human C-mannosyltransferases, and to develop an in vitro C-mannosyltransferase activity assay. Using radiolabelling methodoly, C-mannosyltransferase activity in pig liver was shown for the first time.

Carbohydrates are broken down by highly specific enzymes which are able to hydrolyse the glycosidic bond between glyco-conjugates. The inability to hydrolyse glycans can lead to some of the most common genetically based syndromes in man, including lactose intolerance, and also to lysosomal storage disorders. By understanding the mechanism of action for glycoside hydrolases, new treatments can be developed. The work undertaken here investigated three bacterial α-galactosidases from the GH36 family and their interactions with novel inhibitors. The structural and kinetic analysis aided understanding of the conformational pathway of the hydrolysis reaction catalysed by these α-galactosidases and allowed confirmation of the catalytic nucleophile for two of the enzymes.

Date of Award	21 Jun 2017
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Tracey Gloster (Supervisor)