Structural-functional characterization of ligand binding to the active site and allosteric site of UDP-Galactopyranose mutase from M. Tuberculosis
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The enzyme UDP-galactopyranose mutase (UGM) is essential for the biosynthesis of the cell wall of Mycobacterium tuberculosis, the causative agent for tuberculosis (TB). MtUGM is a member of the pyranose-furanose mutases and is involved in the production of UDP-galactofuranose (UDP-Galf). Galf is not observed in mammals and therefore inhibitors of UGM could be used as a new class of antimycobacterial agents. Due to increase of drug resistant bacteria, it is important to design new inhibitors that bind to sites other than the active site of the enzyme, and the inhibitor MS-208 was proposed to bind to a novel allosteric site in MtUGM. This thesis reports the design of mutants of MtUGM, their expression, purification and characterization to probe the binding of MS-208 inhibitor to the allosteric site. Computational docking studies have depicted different binding modes of MS-208 in the allosteric site of these mutants, while inhibitory analysis showed a change in the type of inhibition among tested allosteric site mutants. Additionally, the structures of the R257A and E321A mutants have been determined by X-ray crystallography. The crystallographic results revealed some of the residues that may be involved in positioning the inhibitor in the allosteric site. Additionally, various prokaryotic UGM species were tested for presence of the allosteric site. Next inhibition analysis with MS-208 was performed on UGM from Deinococcus radiodurans. This data shows that MS-208 can inhibit various prokaryotic UGMs. A fluorescence-based competitive binding assay using the fluorophore 8-anilinonaphthalene-1-sulfonic acid (ANS) has been used to probe ligand binding to MtUGM. It was shown that ANS behaves as a competitive inhibitor of MtUGM. ANS was docked and kinetically tested with two allosteric site mutants (Y253A and D322A) and showed competitive behavior against these mutants. These results show that ANS inhibits and binds to the enzyme differently than MS-208. It has been proposed that two strictly conserved active site residues (W166 and R261) regulate active site closure and control binding of the substrate in the productive conformation in prokaryotic UGM. Kinetic, modeling, and crystallization results showed that W166A and R261A mutants of the enzyme are still catalytically active. The crystal structure of MtUGM in the presence of an UDP-F2-Galf substrate analog has been determined for further active site studies. To date, it is only the second available structure of the possible binding mode for the original substrate, UDP-Galf.
DegreeDoctor of Philosophy (Ph.D.)
CommitteePalmer, David; Krol, Ed; Leung, Adelaine; Mueller, Jens
Copyright DateDecember 2020
allosteric site, inhibitors, active site, M. Tuberculosis, fluorinated substrate, UDP, UDP-Galactopyranose, UDP-Galactofuranose, ANS, tuberculosis, crystal structure, MS-208 inhibitor, docking studies, mixed inhibition, competitive inhibition, X-ray crystallography, allosteric site mutants, fluorescence-based competitive binding assay, competitive inhibitor,