| Carbohydrate polymers are the most abundant organic compounds on earth. The crucial functions of polysaccharides depend on length and chemical structure, yet glycosyltransferases regulate these properties without using a template. Moreover, inhibitors of polysaccharide formation could provide probes of carbohydrate function as well as lead therapeutic compounds for polysaccharides that function in pathogenesis. Here, I present studies on the mechanism and inhibition of biosynthesis of the galactan, a critical polymer of galactofuranose residues found in the mycobacterial cell wall.;Glycoconjugates that contain galactofuranose, the rare five-membered ring form of galactose, are essential for virulence or viability of various pathogens, including the causative agent of tuberculosis. The biosynthetic precursor of galactofuranose residues, UDP-galactofuranose, is produced from UDP-galactopyranose by the flavoenzyme UDP-galactopyranose mutase (UGM, encoded by glf or GLF). I discovered inhibitors of mycobacterial UGM using a high-throughput screen of commercially available small-molecule libraries. From a directed, synthetic library of 2-aminothiazole derivatives, I identified UGM inhibitors that prevent mycobacterial growth. These studies indicate that UGM is a therapeutic target for tuberculosis. We anticipate that the 2-aminothiazole scaffold can afford compounds to combat diseases caused by galactofuranose-containing organisms and serve as probes of the functions of galactofuranose-containing glycoconjugates.;I also examined how the galactofuranosyltransferase GlfT2 mediates polymerization to form the galactan. Using synthetic substrates, I found that G1fT2 is processive, and my data indicate that it controls galactan length by tethering the growing polymer at both ends. The length of the resulting polymer depends on the binding strength of the tether. Tethering represents a potentially general mechanism for length control in polysaccharide biosynthesis.;Lastly, I investigated whether GlfT2, which forms alternating beta-(1→6) and beta-(1→5) glycosidic linkages, uses one or two active sites for dual regioselectivity. Sequence analysis reveals that highly conserved sequences fall within the only glycosyltransferase domain of GlfT2. Homology modeling suggested that these motifs play distinct roles within this catalytic site. Using site-directed mutagenesis, one DDX sequence was found to be critical for bifunctionality. These results indicate that GlfT2 mediates bifunctional catalysis with one active site. |
