Abstract
Abstract Glucoamylase (GA) from Rhizopus oryzae, a glycoside hydrolase, consists of two functional domains, an N-terminal starch-binding domain (SBD) and a C-terminal catalytic domain. The SBD is classified as a member of the carbohydrate-binding module (CBM) family 21 and possesses a high binding affinity toward raw starch. SBD promotes interaction between the ligand and GA, and increases local ligand concentration at the active site of the catalytic domain. In terms of ligand-binding, the stacking of aromatic residues against the sugar rings of polysaccharides/oligosaccharides acts as a key determinant of overall affinity and specificity, and direct hydrogen bonding between the hydroxyl groups of carbohydrates and polar residues in the binding sites of CBMs is also characteristic. Based on the exact three dimensional structure of the SBD in the presence of a cyclic ligand β-cyclodextrin (βCD) determined by NMR and X-ray crystallography, potential ligand-binding residues (Asn29, Tyr32, Lys34, Trp47, Asn50, Phe58, Tyr67, Glu68, Tyr83, Tyr94, Asn96 and Asn101) have been suggested. Here site-directed mutagenesis, circular dichroism (CD), and quantitative binding assays were performed to characterize important hydrophobic stacking interactions and direct hydrogen bonds between our type B CBM and polysaccharides/oligosaccharides. Our results reveal that aromatic and polar residues play distinct roles in ligand-binding. The former act as the major ligand-binding moieties, whereas the latter assist in forcing starch strands twisting apart to expose more ligand surface for more SBD binding. Interestingly, all key aromatic residues characterized in our SBD have counterparts in the other SBD-containing families including CBM20, CBM25, CBM26, CBM34, CBM41, CBM45, and CBM48. Taken together, our results indicate that although only extremely low sequence homology exists among these SBD-containing CBM families, functionally important residues have been well conserved through evolution.