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Chemical Studies Exploring the Specificity of Bacterial Furanoside Biosynthesis Open Access


Other title
Furanoside biosynthesis
furanose sugar nucleotide
UDP-galactopyranose mutase
Type of item
Degree grantor
University of Alberta
Author or creator
Poulin, Myles B.
Supervisor and department
Lowary, Todd (Chemistry)
Examining committee member and department
Campbell, Robert (Chemistry)
Schang, Luis (Biochemistry)
Loppnow, Glen (Chemistry)
Liu, Hung-Wen (Chemistry and Biochemistry, The University of Texas Austin)
Department of Chemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
Mammalian glycoconjugates are composed exclusively of sugar in the thermodynamically favorable pyranose ring form; on the other hand, sugars in the five-membered furanose ring form are widespread in many bacteria, fungi, and protozoan pathogens. This makes the enzymes involved in furanoside biosynthesis a potential selective drug target for anti-microbial therapeutics. However, many questions regarding the binding interactions that occur in the active sites of these enzymes still remain unanswered. In particular, protein structural motifs involved in substrate discrimination remain poorly understood. Galactofuranose, the most common hexofuranose sugar, is biosynthesized by an enzyme known as UDP-galactopyranose mutase (UGM). In contrast, the role of similar enzymes involved in the biosynthesis of various other furanose sugars has not been established. Of particular interest to this thesis are the furanose sugars found in the capsular polysaccharides of Campylobacter jejuni; specifically the 2-acetamido-2-deoxy-D-galactofuranose residue produced by C. jejuni serotype HS:2 and the D-fucofuranose, 6-deoxy-L-altrofuranose, L-arabinofuranose, and 6-deoxy-D-altro-heptofuranose produced by C. jejuni serotype HS:41 whose biosynthesis remain unexplored. Herein, we examine the activity and specificity of the pyranose–furanose mutase enzymes responsible for the biosynthesis of these sugars. Using synthetic substrate analogs and molecular biology techniques, we have also evaluated specific binding interactions responsible for the substrate specificity of these enzymes. In addition to the pyranose–furanose mutase, furanosyltransferase enzymes are also involved in furanoside biosynthesis. Specifically, the mycobacterial cell wall galactan, composed of alternating β-(1→5) and β-(1→6) galactofuranosyl residues, is assembled by the action of two bifunctional galactofuranosyltransferases, GlfT1 and GlfT2. The second, GlfT2, adds the third and subsequent Galf residues using a single active site to carry out both the formation of β-D-Galf-(1→5)-β-D-Galf and β-D-Galf-(1→6)-β-D-Galf linkages. Previous work has largely focused on the specificity of the acceptor species with little known with regards to the binding and specificity of the UDP-D-Galf donor. Herein, we have used a range of synthetic UDP-D-Galf analogs to probe the specificity of GlfT2, and site-directed mutagenesis to explore the mechanism of alternating β-(1→5) and β-(1→6)-GlfT activity. Together, these observations provide insight into specific protein–carbohydrate interactions in GlfT2 and may facilitate the design of future inhibitors.
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
Poulin, M. B.; Lowary, T. L. (2010) Methods in Enzymology., 478, 389-411.Poulin, M. B.; Nothaft, H.; Hug, I.; Feldman, M. F.; Szymanski, C. M.; Lowary, T. L. (2010) J. Biol. Chem., 285, 493-501.Poulin, M. B.; Zhou, R.; Lowary, T. L. (2012) Org. Biomol. Chem., 10, 4074-4087.

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