Abstract:The starch-hydrolysing enzyme GA (glucoamylase) from Rhizopus oryzae is a commonly used glycoside hydrolase in industry. It consists of a C-terminal catalytic domain and an Nterminal starch-binding domain, which belong to the CBM21(carbohydrate-binding module, family 21). In the present study, a molecular model of CBM21 from R. oryzae GA (RoGACBM21) was constructed according to PSSC (progressive secondary structure correlation), modified structure-based sequence alignment, and site-directed mutagenesis was used to identify and characterize potential ligand-binding sites. Our model suggests that RoGACBM21 contains two ligand-binding sites, with Tyr32 and Tyr67 grouped into site I, and Trp47, Tyr83 and Tyr93 grouped into site II. The involvement of these aromatic residues has been validated using chemical modification, UV difference spectroscopy studies, and both qualitative and quantitative binding assays on a series of RoGACBM21 mutants. Our results further reveal that binding sites I and II play distinct roles in ligand binding, the former not only is involved in binding insoluble starch, but also facilitates the binding of RoGACBM21 to long-chain soluble polysaccharides,whereas the latter serves as the major binding site mediating the binding of both soluble polysaccharide and insoluble ligands. In the present study we have for the first time demonstrated that the key ligand-binding residues of RoGACBM21 can be identified and characterized by a combination of novel bioinformatics methodologies in the absence of resolved threedimensional structural information.