Structural and Biochemical Characterization of a Type II Arabinogalactan Fucosyltransferase 4 from Arabidopsis thaliana

Plant cell walls are made of complex polymeric networks of diverse polysaccharides, proteins, polyphenolics, and glycoproteins called hydroxyproline-rich glycoproteins (HRGPs), like arabinogalactan proteins or AGPs. Approximately 90 to 98% of the mass of AGPs is composed of polysaccharide components, which are synthesized by a group of enzymes known as glycosyltransferases (GTs). One subset of these enzymes comprises a family of fucosyltransferases (FUTs) that transfer fucose (Fuc) residues to the arabinose (Ara) moiety of AGP. Ten FUTs involved in plant cell wall polymer synthesis have been identified in Arabidopsis thaliana, three of which have been characterized: AtFUT1, a xyloglucan-specific FUT, and AtFUT4 and AtFUT6, which are AGP-specific FUTs. In this study, we determined the crystal structure of AtFUT4 to gain insights into its substrate specificity toward GDP-Fuc (donor substrate) and AGP-based oligo- and polysaccharides (acceptor substrates). Through integrated bioinformatics and biochemical analyses, we identified key features that define acceptor- and donor-specificities. We tested two acceptors, AGPs and the pseudo-substrate arabinotetrose, and identified critical residues that contribute to acceptor recognition. In addition to identifying residue-level interactions, our analysis highlights the importance of structural flexibility: tighter binding of GDP-fucose by AtFUT4 likely enables broader acceptor specificity compared with AtFUT1, while also increasing donor hydrolysis in the absence of acceptor. Overall, the study advances understanding of the structure-function relationship of AtFUT4 in AGP fucosylation and provides a foundation for rational engineering of GTs, particularly FUTs, development of inhibitors, studies of cell wall polysaccharide interactions, and exploration of the roles of AtFUT4 in plant physiology.