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Structure and function of glycoside hydrolase enzymes involved in mannose and N-acetylglucosamine processing

Males, Alexandra (2019) Structure and function of glycoside hydrolase enzymes involved in mannose and N-acetylglucosamine processing. PhD thesis, University of York.

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Abstract

The growth in the knowledge of the glycome vastly increased in the 20th century to advance the field of chemical biology. The variety of combinations that can be created from monosaccharide building blocks lead to the diverse functions that carbohydrates possess in the cell and organism. The importance of understanding the chemical structures of carbohydrates was a catalyst to aquiring the complex geometries and distorted substrate conformation analysis of the enzymes performing the reactions. Glycoside hydrolases (GHs) cleave the glycosidic bond between a carbohydrate and another carbohydrate or non-carbohydrate molecule. They are classified into different families according to amino-acid sequence. This thesis focuses on three CAZy families, where activity and mechanisms reflect their C2-substituents. GH47 and GH125 enzymes are α-mannosidases involved in the hydrolysis of α-1,2-linked and α-1,6-linked manno-saccharides, respectively. CAZy family GH84 enzymes cleave N-acetyl β-D-glucosamine (GlcNAc) from serine or threonine residues of proteins. Although each enzyme family catalyses a different reaction with different mechanisms and substrates, mannose and GlcNAc have a common feature. Dominating the reactivity of these enzymes, the substituent at the C2 position shows a preference for the alignment of the anomeric oxygen in an axial position and limits the number of accessible distorted conformations. In the case of manno-active enzymes, the axial disposition of O2 in the preferred 4C1 conformation hinders nucleophilic substitution and so, on-enzyme, mannose favours skew boat and boat conformations that render O2 equatorial. In contrast, the C2 N-acetyl substituent position of GlcNAc allows nucleophilic attack at C1 via a neighbouring group participation mechanism. In this thesis, studying the conformational itinerary of these enzymes and conducting inhibition and activity studies using X-ray crystallography and biophysical techniques provides information on substrate specificity and will inform the design of mechanism-based inhibitors as probes with the potential for therapeutic applications.

Item Type: Thesis (PhD)
Related URLs:
Academic Units: The University of York > Chemistry (York)
Depositing User: Miss Alexandra Males
Date Deposited: 16 Jan 2020 14:04
Last Modified: 16 Jan 2020 14:04
URI: http://etheses.whiterose.ac.uk/id/eprint/25593

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