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Permanent link (DOI): https://doi.org/10.7939/R36688T0W

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Studies of a mycobacterial α-(1→4)-mannosyltransferase involved in 3-O-methyl-mannose polysaccharide biosynthesis Open Access

Descriptions

Other title
Subject/Keyword
Regioselective polymethylation
MMP biosynthesis
α-(1→4)-mannosyltransferase
3-O-methyl-mannose polysaccharide
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Xia, Li
Supervisor and department
Lowary, Todd L. (Chemistry)
Examining committee member and department
Thorson, Jon S. (College of Pharmacy, University of Kentucky)
Gibbs-Davis, Julianne M. (Chemistry)
Clive, Derrick L. J. (Chemistry)
Cairo, Christopher W. (Chemistry)
Xu, Yunjie (Chemistry)
Department
Department of Chemistry
Specialization

Date accepted
2013-01-28T15:26:33Z
Graduation date
2013-06
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Mycobacteria produce a number of unique carbohydrates, the majority of which are found in the cell wall of the organism. In addition to these extracellular glycans, mycobacteria also produce intracellular glycans. One class of them are the 3-O-methy-lmannose polysaccharides (MMPs), whose physiological functions and biosynthesis have not been fully established. An α-(1→4)-mannosyltransferase (ManT), one of the enzymes involved in MMP biosynthesis, incorporates mannose via an unusual α-(1→4)-linkage. However, to date, there is only one report studying this uncommon glycosyltransferase. In this thesis, we sought to expand upon this study to fully characterize the activity, specificity, and identity of ManT. We hypothesized that access to synthetic MMP fragments would lead to a greater understanding of the substrate specificity of ManT. When using synthetic MMP analogs to probe this enzyme, an unexpected activity for ManT was discovered. This enzyme was previously reported to recognize only substrates having a terminal methylated residue. However, we demonstrated that ManT also recognizes substrates without a methyl group on this terminal mannose residue, and with even better affinity. This finding was supported through careful structural elucidation of the isolated enzymatic products with various techniques including glycosidase digestion, Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) and Nuclear Magnetic Resonance (NMR) spectroscopy, and a comparison of the kinetic parameters of two representative tetrasaccharide substrates. To further our understanding of MMP biosynthesis we sought to obtain pure ManT enzyme. Our first attempt to purify ManT using synthetic MMP-functionalized affinity chromatography was not successful. Therefore, a bioinformatic approach was used. Following this approach, we identified a putative ManT gene from M. smegmatis, and expressed this gene recombinantly in E. coli. The activity of the gene product was found to be identical to the native ManT from M. smegmatis. This will facilitate further studies of MMP biosynthesis. In the course of synthesizing MMP analogs for these studies, we developed a four-step methodology to quickly install several methyl groups directly on oligosaccharides. The key reaction involves n-Bu2SnCl2-mediated simultaneous activation of multiple cis-diols. By tuning protecting groups on the substrates, we were able to functionalize multiple cis-diols in a consistent and highly regioselective manner.
Language
English
DOI
doi:10.7939/R36688T0W
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.
Citation for previous publication
Xia, L.; Zheng, R. B.; Lowary, T. L. ChemBioChem 2012, 13, 1139-1151.

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