Abstract
Rhizopus oryzae glucoamylase is composed of an N-terminal starch binding domain (RoSBD, residues 26-131) and a C-terminal catalytic domain (residues 168-604) connected by an O-glycosylated linker (residues 132-167). RoSBD is categorized to carbohydrate binding modules (CBMs) family 21, and has been demonstrated to effectively adsorb onto raw starch and other soluble oligosaccharides. Three dimensional structures of RoSBD in the presence of a cyclic ligand β-cyclodextrin (βCD) or a linear maltoheptaose (G7) have been determined by NMR and X-ray crystallography. RoSBD possesses two ligand binding sites, residues Trp47, Tyr83, Tyr93, and Tyr94 constitute binding site I, and residues Tyr32, Phe58, and Tyr67 form binding site II. Hydrophobic interaction between the sugar rings of ligands and aromatic residues of RoSBD act as a key determinant of overall binding affinity and specificity. Here isothermal titration calorimetry (ITC), fluorescence spectrophotometry and depletion isotherm have been used to identify binding affinities of various glycans containing cyclic and linear linkages with special focus on the deterministic factors such as length, glycosidic linkage, ring size, and solubility of polysaccharides. Micromolar range binding affinities of different ligands are observed. In addition, to distinguish the affinity of two ligand binding sites, key binding residue of two sites, Trp47 in site I and Tyr32, Phe58 in site II are specifically mutated. Interestingly, Tyr32 mutant (Y32A) shows over 10 fold weaker binding affinity than wild-type RoSBD, strongly suggesting that Tyr32 plays a more important role in binding soluble glycans. X-ray crystallography results indicate that the side chain of Tyr32 significantly flips over to form a ligand binding clamp with Phe58 upon binding to either βCD or G7. In addition, unique polyN loops in binding site I form another ligand binding clamp to cooperatively interact with ligand through hydrogen bonding. The two binding sites of RoSBD appear to play distinct roles in ligand binding. Trp47 along with polyN loops initially bind to insoluble starch strands and force starch to twist apart to expose extensive surface for solvent access. In addition, Tyr32 and Phe58 in binding site II form a ligand binding clamp to coordinately bind to soluble starch with high binding affinity. Finally, bioinformatic analysis demonstrates binding properties, correlation of two ligand binding sites, and conservation of binding residues of RoSBD, which in turn leads to more understanding in structure-function relationship of SBD and CBM.