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Structural analysis of UDP-N-acetylgalactopyranose mutase from Campylobacter jejuni 11168

dc.contributor.advisorSanders, David A.en_US
dc.contributor.committeeMemberMoore, Stanleyen_US
dc.contributor.committeeMemberFodje, Michelen_US
dc.contributor.committeeMemberYang, Jianen_US
dc.creatorProtsko, Carlaen_US 2012en_US
dc.description.abstractUDP-galactopyranose mutase (EC; UGM), the product of the glf gene, is an enzyme that catalyzes the conversion of uridine diphosphate galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UGM activity is found in bacteria, parasites and fungi, however is absent in higher eukaryotes. This enzyme is essential for the viability of many pathogenic organisms, such as Mycobacterium tuberculosis and Escherichia coli, due to the broad distribution of Galf in crucial structures such as the cell wall or capsular polysaccharide. Not surprisingly, galactofuranose biosynthesis has become an attractive antimicrobial target due to the absence of these sugars in higher eukaryotes. The UGM homologue, UDP-Nacetylgalactopyranose mutase (UNGM), was identified in Campylobacter jejuni 11168, encoded for by the cj1439c gene. UNGM is known to function as a bifunctional mutase, which catalyzes the reversible ring contraction between the pyranose-furanose forms of UDPgalactose (UDP-Gal) and UDP-N-acetylgalactosamine (UDP-GalNAc). UNGM is essential for the virulence of C. jejuni, due to the incorporation of UDP-N-acetylgalactofuranose into the capsular polysaccharide. We report the first structure of UNGM determined by X-ray crystallography, to a resolution of 1.9 Å. Analysis of the dimeric, holoenzyme structure of UNGM has identified that the cofactor flavin adenine dinucleotide is bound within each monomer of the enzyme. Comparative analysis with UGM homologues has confirmed the conserved active site residues involved in the binding of various substrates. Docking studies suggest that UNGM binds its natural substrates in a productive binding mode for catalysis with the flavin cofactor, which is consistent with the proposed mechanism for UNGM. The mobile loops are essential for substrate binding, and we have identified that the conserved arginine residue, Arg169, and the neighboring Arg168, function to stabilize the diphosphate region of UDP, although not concurrently. The non-conserved arginine residue, Arg168, appeared to favor the stabilization of N-acetylated sugars, which is in agreement with the enzyme’s higher binding affinity for UDP-GalNAc over UDP-Gal by a factor of 0.9. We have also identified that the active site Arg59 exists in two conformations in the structure of UNGM, with one conformation directed toward the active site. Arg59 is 2.5 to 3.0 Å from the acetamido moiety of GalNAc, which is favorable for stabilization and is believed to confer specificity for this substrate.en_US
dc.subjectUDP-N-acetylgalactopyranose mutaseen_US
dc.subjectdual specificityen_US
dc.subjectcapsular polysaccharideen_US
dc.titleStructural analysis of UDP-N-acetylgalactopyranose mutase from Campylobacter jejuni 11168en_US
dc.type.materialtexten_US of Saskatchewanen_US of Science (M.Sc.)en_US


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