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  • Two unique ligand-binding clamps of Rhizopus oryzae starch binding domain for helical structure disruption of amylose.

Two unique ligand-binding clamps of Rhizopus oryzae starch binding domain for helical structure disruption of amylose.

PloS one (2012-07-21)
Ting-Ying Jiang, Yuan-Pei Ci, Wei-I Chou, Yuan-Chuan Lee, Yuh-Ju Sun, Wei-Yao Chou, Kun-Mou Li, Margaret Dah-Tsyr Chang
要旨

The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD) has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21) members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs.

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Sigma-Aldrich
アミログルコシダーゼ from Aspergillus niger, lyophilized powder, 30-60 units/mg protein (biuret), ≤0.02% glucose
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アミログルコシダーゼ from Aspergillus niger, lyophilized, powder, ~70 U/mg
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アミログルコシダーゼ from Aspergillus niger, powder, white, ~120 U/mg
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アミログルコシダーゼ from Aspergillus niger, ammonium sulfate suspension, ≥40 units/mg protein
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アミログルコシダーゼ from Aspergillus niger, ≥260 U/mL, aqueous solution
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アミログルコシダーゼ from Aspergillus niger, Isoelectric focusing marker, pI 3.6
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アミログルコシダーゼ from Rhizopus sp., ≥40,000 units/g solid