Journal
BIOCHEMICAL JOURNAL
Volume 416, Issue -, Pages 27-36Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20080580
Keywords
carbohydrate-binding module; beta-cyclodextrin; maltoheptaose; Rhizopus oryzae glucoamylase; starch-binding domain
Categories
Funding
- National Science Council of Taiwan [NSC 96-2311-B-007-014, NTHU 96N2424E1, NSC 96-2627-B-007-003]
- Council of Agriculture
- National Science and Technology Programme for Agricultural Biotechnology [97AS-1.2.1.-ST-a5]
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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-beta CD) 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.
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