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Structural and functional aspects of pyranose-furanose mutases



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Pyranose-Furanose mutases are enzymes that catalyze the isomerization of six-membered pyranose and five-membered furanose forms of a nucleotide-based sugar. In this research, the substrate binding site of three different mutases were investigated; UDP-galactopyranose mutase (UGM), GDP-altro-heptopyranose mutase (GaHM) and UDP-arabinopyranose mutase (UAM). Both UGM and UAM use a UDP-based sugar as the substrate but require different cofactors, flavin adenine dinucleotide (FAD) and Mn2+ respectively, to function. UGM and GaHM use the same cofactor (FAD), but the latter prefers to work with a GDP-based sugar. In this thesis, studies have been conducted on these three mutases using a variety of tools, such as X-ray crystallography, protein modeling, site-directed mutagenesis and kinetic assays, to understand how these enzymes bind their respective substrates. Among these three mutases, UGM is the best-studied enzyme and is a validated drug target in Mycobacteria. Despite this, the structural role of some active site residues in substrate binding is not clearly understood. Deinococcus radiodurans UGM (DrUGM) mutants of active site residues Trp184, Arg364, His88, and Asn372 were prepared and evaluated using kinetic and docking studies. The results suggested that these residues are vital to the positioning of UDP-galactopyranose under FAD in a productive conformation, for maximum enzyme efficiency. Inhibition studies, using the inhibitor MS-208, were performed on Mycobacterium tuberculosis UGM (MtUGM). Kinetic assays indicated that MS-208 is a mixed-type inhibitor of MtUGM. In this study, the crystal structures of Campylobacter jejuni GaHM (CjGaHM) with a substrate mimic GDP-mannose were solved, allowing for a comparison of GaHM and UGM substrate binding sites. The results highlighted the alterations undergone by CjGaHM to accommodate a GDP-based substrate in the active site. A preliminary model of UAM was built based on the protein sequence of Oryza sativa UAM1 (OsUAM1) using the protein structure modeling servers I-TASSER and GalaxyWEB. The models suggested that, unlike the catalytic role played by the FAD cofactor in UGM and GaHM, the role of the Mn2+ cofactor in UAM could be to aid the stabilization of the negative charge of the substrate diphosphate. Furthermore, experiments with mutants of OsUAM1 have helped identify residues that may bind the metal cofactor.



Pyranose-Furanose mutases, UGM, UAM, GaHM



Doctor of Philosophy (Ph.D.)







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