| Glycans mediate a wide range of biological processes. They are a diverse and abundant class of molecules, particularly notable for their complexity. Assembly of glycan chains takes place through a template-independent process mediated by enzymes termed glycosyltransferases. The oligosaccharide portion of glycosyltransferase acceptors are frequently linked via a pyrophosphate bridge to a long lipid carrier, and all components are recognized by the enzyme. Deciphering the enzymes' function in vitro is complicated by the lability and amphiphilic nature of these glycan intermediates, and the challenges in accessing them synthetically. I sought to address this limitation while investigating the galactofuranosyltransferases that mediate galactan biosynthesis. Galactan is a glycan polymer consisting of 20--40 galactofuranose (Galƒ) residues, built on a pyrophosphoryl-linked carrier lipid prior to installation onto the cell wall peptidoglycan of mycobacteria.;The galactofuranosyltransferase GlfT1 is hypothesized to prime galactan assembly by elongating a lipid-linked disaccharide pyrophosphate with 2--3 Galƒ residues. The polymerase GlfT2 builds the full galactan chain from this oligosaccharide. Efforts to characterize GlfT1 using O-alkyl disaccharide acceptor analogs have failed, but it was unclear whether this resulted from instability of the enzyme in vitro or lack of suitable acceptors. I hypothesized that GlfT1 is restrictive for disaccharide acceptor analogs bearing a pyrophosphate, or analog thereof. I stabilized the putative GlfT1 acceptor substrate by replacing the pyrophosphate group with a phosphonophosphate.;Using synthetic acceptor substrate surrogates, I discovered that GlfT1 elongates disaccharide acceptors by 2--3 Galƒ residues in vitro, generating a specific oligosaccharide pattern. In generating the +3 Galƒ oligosaccharide, GlfT1 sets the register for the alternating beta-(1,5) and beta-(1,6) linkage pattern observed in endogenous galactan. Chain termination experiments utilizing deoxygenated UDP-Galf donors and NMR spectroscopy confirmed the Galƒ-beta-(1,6)-Galƒ-beta-(1,5)-Galƒ pattern on the non-reducing end of the lipid-linked phosphonophosphate. Finally, I determined that an acceptor substrate bearing the Galƒ-beta-(1,5)-Rha-alpha-(1,3)-GlcNAc trisaccharide overrides the enzyme's requirement for a pyrophosphate-containing substrate. Using this acceptor, I concluded that GlfT1 produces oligosaccharides that can be extended by the polymerase GlfT2 to generate synthetic galactan polymers of endogenous length. |
