Premium
Defining Starch Binding by Glucan Phosphatases
Author(s) -
Auger Kyle,
Raththagala Madushi,
Wilkens Casper,
Svensson Birte,
Gentry Matthew
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.lb205
Subject(s) - amylopectin , glucan , biochemistry , amylose , phosphatase , affinity electrophoresis , chemistry , starch , carbohydrate binding module , enzyme , glycoside hydrolase , affinity chromatography
Starch is a vital energy molecule in plants that has a wide variety of uses in industry, such as feedstock for biomaterial processing and biofuel production. Plants employ a three enzyme cyclic process utilizing kinases, amylases, and phosphatases to degrade starch in a diurnal manner. Starch is comprised of the branched glucan amylopectin and the more linear glucan amylose. Our lab has determined the first structures of these glucan phosphatases and we have defined their enzymatic action. Despite this progress, we lacked a means to quickly and efficiently quantify starch binding to glucan phosphatases. The main objective of this study was to quantify the binding affinity of different enzymes that are involved in this cyclic process. We established a protocol to quickly, reproducibly, and quantitatively measure the binding of the enzymes to glucans utilizing Affinity Gel Electrophoresis (AGE) and Surface Plasmon Resonance (SPR). The results show that the glucan phosphatases possess differing abilities to bind to different glucan substrates. One glucan phosphatase possesses a 50 fold higher affinity for amylopectin than the other, while it only possessed a 3 fold higher affinity for amylose. Both glucan phosphatases showed similar affinities for the short oligosaccharide β‐cyclodextrin. We performed structure‐guided mutagenesis to define the mechanism of these differences. We found that the carbohydrate binding module (CBM) domain provided a stronger binding affinity compared to surface binding sites (SBSs).