A CBM20 low‐affinity starch‐binding domain from glucan, water dikinase

Abstract

Edited by Michael R. Sussman Keywords:Bioimaging Carbohydrate-binding module 20 Glucan, water dikinase Starch-binding domain Surface plasmon resonance a b s t r a c tThe family 20 carbohydrate-binding module (CBM20) of the Arabidopsis starch phosphorylator glucan, water dikinase 3 (GWD3) was heterologously produced and its properties were compared to the CBM20 from a fungal glucoamylase (GA). The GWD3 CBM20 has 50-fold lower affinity for cyclodextrins than that from GA. Homology modelling identified possible structural elements responsible for this weak binding of the intracellular CBM20. Differential binding of fluorescein-labelled GWD3 and GA modules to starch granules in vitro was demonstrated by confocal laser scanning microscopy and yellow fluorescent protein-tagged GWD3 CBM20 expressed in tobacco confirmed binding to starch granules in planta.

A Putative Phosphatase, LSF1, Is Required for Normal Starch Turnover in Arabidopsis Leaves

“…The nonuniform distribution of fluorescence from the LSF1-GFP fusion protein strongly resembled that from a chloroplast-targeted yellow fluorescent protein fusion of the starch-binding domain (a family 20 carbohydrate-binding module) of PWD. The PWD starch-binding domain also binds to starch granules in vitro (Christiansen et al, 2009). …”

New Insight into Structure/Function Relationships in Plant .ALPHA.-Amylase Family GH13 Members

“…14) Most remarkably, while CBM20s in amylolytic enzymes confer binding affinities yielding dissociation constants for the starch mimic β CD in the low μM range, 29,30) CBM20 from a regulatory enzyme was very recently prepared and shown to have weaker affinity and Kd in the low mM range. 15) This is proposed to facilitate binding and release of the regulatory enzyme and a structural basis for the difference in affinity is indicated by distinct architectural features of the two SBD binding sites and roles of pivotal amino acid residues in binding. 15) This functional difference, which has been attributed to a deletion in the flexible loop implicated in ligand binding at the higher affinity binding site two in the canonical glucoamylase starch The tree illustrates the clustering of SBDs from CBM20 based on the catalytic modules associated with them.…”

“…15) This is proposed to facilitate binding and release of the regulatory enzyme and a structural basis for the difference in affinity is indicated by distinct architectural features of the two SBD binding sites and roles of pivotal amino acid residues in binding. 15) This functional difference, which has been attributed to a deletion in the flexible loop implicated in ligand binding at the higher affinity binding site two in the canonical glucoamylase starch The tree illustrates the clustering of SBDs from CBM20 based on the catalytic modules associated with them. Hence, SBDs from GH13, GH14 (both of bacterial origin), GH15 (of fungal origin) and "Plant" (these are of GH77) cluster on separate branches, whereas the SBDs occurring with starch or glycogen metabolic regulatory activities, e.g.…”

“…Currently 9 different CBMs families have specificity for starch granules or other α glucan polymers such as glycogen. 14,15) Very recently a subgroup of CBM20 from regulatory enzymes, was revealed in the starch phosphorylator phosphoglucan, water dikinase 3 from Arabidopsis thaliana to have exceptionally weak carbohydrate affinity. 15) GH13 includes both many enzymes with activity towards α 1,4 linkages in α glucans and enzymes that specifically catalyse attack on α 1,6 branch points in amylopectin or related poly and oligosaccharides.…”

“…14,15) Very recently a subgroup of CBM20 from regulatory enzymes, was revealed in the starch phosphorylator phosphoglucan, water dikinase 3 from Arabidopsis thaliana to have exceptionally weak carbohydrate affinity. 15) GH13 includes both many enzymes with activity towards α 1,4 linkages in α glucans and enzymes that specifically catalyse attack on α 1,6 branch points in amylopectin or related poly and oligosaccharides. 16,17) One of these debranching enzymes, limit dextrinase (LD), has been cloned from barley and produced recombinantly in high yield with excellent specific activity 18 21) using Pichia pastoris as host.…”