title: Crystal structures of the starch-binding domain from Rhizopus oryzae glucoamylase reveal a polysaccharide-binding path.
abstract: GA (glucoamylase) hydrolyses starch and polysaccharides to beta-D-glucose. RoGA (Rhizopus oryzae GA) consists of two functional domains, an N-terminal SBD (starch-binding domain) and a C-terminal catalytic domain, which are connected by an O-glycosylated linker. In the present study, the crystal structures of the SBD from RoGA (RoGACBM21) and the complexes with beta-cyclodextrin (SBD-betaCD) and maltoheptaose (SBD-G7) were determined. Two carbohydrate binding sites, I (Trp(47)) and II (Tyr(32)), were resolved and their binding was co-operative. Besides the hydrophobic interaction, two unique polyN loops comprising consecutive asparagine residues also participate in the sugar binding. A conformational change in Tyr(32) was observed between unliganded and liganded SBDs. To elucidate the mechanism of polysaccharide binding, a number of mutants were constructed and characterized by a quantitative binding isotherm and Scatchard analysis. A possible binding path for long-chain polysaccharides in RoGACBM21 was proposed.
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan.
authors.forename: Jung-Yu
authors.initials: JY
authors.lastname: Tung
authors.forename: Margaret Dah-Tsyr
authors.initials: MD
authors.lastname: Chang
authors.forename: Wei-I
authors.initials: WI
authors.lastname: Chou
authors.forename: Yen-Yi
authors.initials: YY
authors.lastname: Liu
authors.forename: Yi-Hung
authors.initials: YH
authors.lastname: Yeh
authors.forename: Fan-Yu
authors.initials: FY
authors.lastname: Chang
authors.forename: Shu-Chuan
authors.initials: SC
authors.lastname: Lin
authors.forename: Zhen-Liang
authors.initials: ZL
authors.lastname: Qiu
authors.forename: Yuh-Ju
authors.initials: YJ
authors.lastname: Sun
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012233:Rhizopus / Q000201:enzymology*; D016415:Sequence Alignment; D013213:Starch / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000378:metabolism
chemical_list: D047392:beta-Cyclodextrins; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase; C031215:betadex