Abstract
Rhamnose is a natural sugar found in glycoproteins and structural polysaccharides of plants, fungi, and bacteria. Its incorporation into glycoconjugates is mediated by rhamnosyltransferases (RHTs), key enzymes for biomolecular stability and function. While rhamnose biosynthesis has been studied in certain fungal genera, the evolutionary history and distribution of RHTs across the fungal kingdom remain largely unknown. In this study, 351 fungal species were found to encode putative RHTs. Phylogenetic and structural analyses revealed conserved patterns and similarities with previously characterized RHTs. Molecular docking predicted a high affinity of these proteins for UDP-L-rhamnose, and in silico mutagenesis identified key residues potentially involved in substrate binding. Carbohydrate profiling confirmed the presence of rhamnose in the cell walls of multiple fungi, including Aspergillus, Madurella, Metarhizium, and Trichoderma species. Enzymatic assays further supported rhamnose transfer activity. These findings provide the first comprehensive in silico characterization of fungal RHTs, uncovering conserved sequence motifs despite overall diversity, which may be linked to functional adaptation in different fungal lineages.