Substrate binding and catalytic mechanism of UDP-α-<scp>D-</scp>galactofuranose: β-galactofuranoside β-(1→5)-galactofuranosyltransferase GfsA

UDP-α-D-galactofuranose (UDP-Galf): β-galactofuranoside β-(1→5)-galactofuranosyltransferase, known as GfsA, is essential in synthesizing β-(1→5)-galactofuranosyl oligosaccharides that are incorporated into the cell wall of pathogenic fungi. This study analyzed the structure and function of GfsA from <i>Aspergillus fumigatus</i>. To provide crucial insights into the catalytic mechanism and substrate recognition, the complex structure was elucidated with manganese (Mn²⁺), a donor substrate product (UDP), and an acceptor sugar molecule (β-galactofuranose). In addition to the typical GT-A fold domain, GfsA has a unique domain formed by the N and C termini. The former interacts with the GT-A of another GfsA, forming a dimer. The active center that contains Mn²⁺, UDP, and galactofuranose forms a groove structure that is highly conserved in the GfsA of Pezizomycotina fungi. Enzymatic assays using site-directed mutants were conducted to determine the roles of specific active-site residues in the enzymatic activity of GfsA. The predicted enzyme-substrate complex model containing UDP-Galf characterized a specific β-galactofuranosyltransfer mechanism to the 5'-OH of β-galactofuranose. Overall, the structure of GfsA in pathogenic fungi provides insights into the complex glycan biosynthetic processes of fungal pathogenesis and may inform the development of novel antifungal therapies.