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Permanent link (DOI): https://doi.org/10.7939/R33S3V

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Studies of glycosyltransferases involved in mycobacterial cell wall biosynthesis Open Access

Descriptions

Other title
Subject/Keyword
mycobacterial cell wall
arabinogalactan
mannosyltransferase
galactofuranosyltransferase
lipoarabinomannan
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Tam, Pui Hang
Supervisor and department
Todd L. Lowary (Chemistry)
Examining committee member and department
Derrick L. J. Clive (Chemistry)
John S. Klassen (Chemistry)
David L. Jakeman (Chemistry, University of Dalhousie)
Robert E. Campbell (Chemistry)
Michael N. James (Biochemistry)
Department
Department of Chemistry
Specialization

Date accepted
2009-09-16T20:24:57Z
Graduation date
2009-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Lipoarabinomannan (LAM) and the mycolyl-arabinogalactan (mAG) complex are two major entities found in the cell wall of Mycobacterium tuberculosis, the bacterium that causes tuberculosis in humans. Given their important roles in the viability and virulence of the pathogen, enzymes involved in these pathways represent a rich source of potential therapeutic drug targets. As fundamental understanding of substrate–enzyme interactions is often essential in the drug discovery process, the purpose of this study was to investigate the substrate specificities of an α-(1→6)-mannosyltransferase (ManT) and a β-(1→5,6)-galactofuranosyltransferase (GlfT2), two key enzymes involved in the biosynthesis of LAM and mAG, respectively. Although the ManT activity had been detected using an established radioactive assay, its substrate specificity remained poorly defined. The current study focused on the design, synthesis and evaluation of acceptor substrate analogs of ManT. Among those analogs prepared were those containing methoxy-, hydrogen-, and amino-substituted carbohydrate residues as well as epimeric derivatives. A homologous series of oxygen- and sulfur-linked mannosides were also prepared. Evaluation of these analogs revealed the steric requirements and hydrogen bonding interactions of the enzyme, and the effect of acceptor length on mannosyltransferase activity. Also, these results provided additional insight into the role of ManTs and allowed the current proposed pathway of LAM to be further revised. Another objective of the current study was to understand how GlfT2 catalyzes the alternating β-(1→5) and β-(1→6)-galactofuranosyl transfers in a single active site. A panel of mono- and dideoxy trisaccharide derivatives was synthesized, in which hydroxyl groups at either or both C-5 and C-6 positions on the sugar residues at the reducing ends were selectively removed. Biological evaluation of these analogs using a spectrophotometric assay, and structural analysis of some of the enzymatic products, showed that the removal of the hydroxyl group(s) in the acceptors appeared to have no dramatic effect on either GlfT2 activity or the regioselectivity of its galactosylation. These results suggest that groups other than the C-5 and C-6 hydroxyl groups of the acceptors are more critical for the enzyme catalysis. The identification of these key elements would be the further objective of this project. The results from these fundamental studies provide important information about how these enzymes interact with their substrates at the molecular level. More importantly, this work will serve as the basis for the further design of potential inhibitors, which are potential lead compounds for novel therapeutic agents that are active against tuberculosis.
Language
English
DOI
doi:10.7939/R33S3V
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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