Abstract
Glucoamylase hydrolyzes starch and polysaccharides to β-d-glucose. Rhizopus oryzae glucoamylase (RoGA) consists of two functional domains, an N-terminal starch binding domain (SBD) and a C-terminal catalytic domain and these two domains are connected by an O-glycosylated linker. The starch-binding domain of RoGA belongs to carbohydrate-binding module 21(CBM21). Two crystal structures of the RoGACBM21 complexes, with a cyclic carbohydrate, β-cyclodextrin (RoGACBM21-βCD), and with a linear carbohydrate, maltoheptaose (RoGACBM21-G7), were determined at 1.8 and 2.3□ respectively. The overall structures belong to a β-sandwich fold with an immunoglobulin-like structure. Two carbohydrate-binding sites were observed. Site I is created by several conserved aromatic residues, Trp47, Tyr83, and Tyr94, which form a broad, flat, and firm hydrophobic binding surface. Site II is built up by Tyr32 and Phe58, which produce a protruded and narrow binding environment. Besides, some hydrophilic residues which are Asn50, Asn96, Asn101 in Site I and Asn29, Glu68, Lys34 in Site II also participate in carbohydrates binding. Liganded and unliganded RoGACBM21s reveal similar overall structures, the major structural difference was found in loop regions and in or near the two carbohydrate-binding sites. Site I undergoes a bigger conformational change than that in site II upon the carbohydrate binding. The overall structure and ligand binding mode of AnGACBM20-βCD complex is similar to RoGACBM21-βCD but the orientations of the βCD at site I and II are different. As for electrostatic potential of RoGACBM21-βCD, it appears a more hydrophobic surface than other surfaces throughout the structure and its opposite side has positive and negative charges separately. Therefore, we can make a suggestion that once a SBD molecule binds to the starch granule surface, it may attract more SBD molecules onto the surface through electrostatic complementation. In conclusion, from electrostatic potential of RoGACBM21-βCD and the ligand binding residues, we could propose a possible role of SBD participating in glucoamylase hydrolysis on the starch granular surface.