Unusual carbohydrate biosynthesis : studies of the flavin-dependent isomerase UGM, the radical S-adenosyl-L-methionine enzyme DesII, and the biosynthesis of herbicidins

Bacteria produce a great variety of unusual carbohydrates that are often found as parts of their cell surfaces or secondary metabolites. These components are crucial for their survival to resist environmental stress or confer bioactivities against competitors. A thorough understanding of their biosynthetic pathways and enzymology can expand our knowledge on nature’s synthetic tools, which can further facilitate the engineering efforts in producing clinically-relevant small molecules. The mechanistic understanding of pathogen-specific enzymes can also serve as the foundation for the drug design. This dissertation describes the investigation into three subjects related to unusual carbohydrate biosynthesis. UDP-galactopyranose mutase (UGM) converts UDP-galactopyranose to UDP-galactofuranose for the cell wall biosynthesis of several pathogens. The catalytic mechanism involves the substrate-flavin adduct formation that is unusual yet crucial for the galactosyl ring contraction. When chemically-synthesized UDP-5F-galactopyranose was incubated with UGM, the formation of substrate-flavin adduct with C5-carbonyl resulting from the elimination of fluoride was demonstrated, substantiating the current mechanistic model. However, the subsequent reaction was diverted to the hydrolysis of the adduct rather than ring contraction. These new findings might aid in the development of mechanism-based inhibitors targeting this enzyme. DesII is a radical S-adenosyl-L-methionine (SAM) enzyme that catalyzes C4-deamination of TDP-4-amino-4, 6-dideoxy-D-glucose in the biosynthesis of desosamine. The studies of 3-deutero-3-fluoro substrate analogues are described to distinguish two mechanistic proposals. The presence of 3-fluoro substituent still permitted the substrate radical formation yet without further conversion; however, the deuterium was found to be freely exchangeable between substrates and SAM. The results argue against the 1,2-migration mechanism in the deamination reaction and suggest that the radical generation is reversible when its forward partitioning is inhibited. Herbicidins are nucleoside antibiotics with unusual tricyclic undecose core, and their biosynthetic origins are initially characterized. The feeding studies revealed that the core is derived from D-glucose and D-ribose. The biosynthetic gene cluster was subsequently identified and verified by its successful expression in a non-producing host. A tentative biosynthetic pathway was then proposed along with the demonstration of the activity of one methyltransferase. This study lays the foundation for the future biosynthetic studies of the eleven-carbon core.Chemistr