Abstract
The plant glucan phosphatases Starch EXcess 4 (SEX4) and Like Sex Four2 (LSF2) apply different starch binding mechanisms. SEX4 contains a carbohydrate binding module, and LSF2 has two surface binding sites (SBSs). We determined KDapp for amylopectin and amylose, and KD for β-cyclodextrin and validated binding site mutants deploying affinity gel electrophoresis (AGE) and surface plasmon resonance. SEX4 has a higher affinity for amylopectin; LSF2 prefers amylose and β-cyclodextrin. SEX4 has 50-fold lower KDapp for amylopectin compared to LSF2. Molecular dynamics simulations and AGE data both support long-distance mutual effects of binding at SBSs and the active site in LSF2.
Keywords:
Like Sex Four2; Starch Excess 4; affinity gel electrophoresis; carbohydrate binding domain; surface binding sites; surface plasmon resonance.
© 2015 Federation of European Biochemical Societies.
Publication types
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Comparative Study
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Amino Acid Substitution
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Amylopectin / chemistry
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Amylopectin / metabolism*
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Amylose / chemistry
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Amylose / metabolism*
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Arabidopsis / enzymology*
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Arabidopsis Proteins / chemistry
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Arabidopsis Proteins / genetics
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Arabidopsis Proteins / metabolism*
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Binding Sites
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Carbohydrate Conformation
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Cytoplasmic Granules / chemistry
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Cytoplasmic Granules / enzymology
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Cytoplasmic Granules / metabolism
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Dual-Specificity Phosphatases / chemistry
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Dual-Specificity Phosphatases / genetics
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Dual-Specificity Phosphatases / metabolism*
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Kinetics
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Models, Molecular*
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Molecular Dynamics Simulation
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Mutation
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Plant Leaves / enzymology*
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Protein Conformation
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Substrate Specificity
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Surface Plasmon Resonance
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beta-Cyclodextrins / chemistry
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beta-Cyclodextrins / metabolism
Substances
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Arabidopsis Proteins
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Recombinant Proteins
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beta-Cyclodextrins
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Amylose
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Amylopectin
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Dual-Specificity Phosphatases
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LSF2 protein, Arabidopsis
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SEX4 protein, Arabidopsis
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betadex
Associated data
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GENBANK/AT3G10940
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GENBANK/AT3G52180
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GENBANK/M73321