Preferential binding of SBD from Arabidopsis thaliana SSIII to polysaccharides: Study of amino acid residues involved

Abstract

SS III (SSIII) has been reported to play a regulatory role in the synthesis of transient starch. SSIII from Arabidopsis thaliana contains 1025 amino acid residues and has an N‐terminal transit peptide for chloroplast localization followed by three in tandem starch‐binding domains (SBDs D1, D2, and D3, residues 22‐591). Its C‐terminal catalytic domain (residues 592–1025) is similar to bacterial glycogen synthase. Binding studies to raw starch and its individual components, AM or AP show that the SBD region binds preferentially to AM, and that the D1 domain is mainly responsible for this selective binding. The D2 domain contains two binding sites which include amino acid residues Y394 (binding site 1) and W366 (binding site 2) acting cooperatively with the D1 domain in the binding process while G335 and W340 have a minor role. In addition, mutations in these residues also affect the kinetic parameters for the polysaccharide substrate of SSIII.

Plant Biotechnology for the Development of Design Starches

“…The adsorption experiments show that the SBD123 region binds preferentially to amylose, and that the D1 domain is mainly responsible for this selective binding. The D2 domain contains two binding sites including amino acid residues Y394 (binding site 1) and W366 (binding site 2) which act in cooperation with the D1 domain in the binding activity while G335 and W340 have a minor role [54]. It is worth mentioning that our work was the first report on the existence of an SBD in a synthesizing enzyme (AtSSIII) and the first experimental evidence of its starch binding capacity.…”

“…C-terminal domains from Lactobacillus manihotivorans -amylase); (v) CBM34, present in the N-terminal domains of neopullulanase, maltogenic amylase and cyclomaltodextrinase; (vi) CBM41, N-terminal SBD, present mostly in bacterial pullulanases; (vii) CBM45, originating from eukaryotic proteins from the plant kingdom (i.e. N-terminal modules of -glucan water dikinases and -amylases); (viii) CBM48, which display glycogen-binding properties (including SBD from the GH13 pullulanase and regulatory modules of mammalian AMP-activated protein kinase); (ix) CBM53, SBD modules from SSIII and (x) CBM58, find in -amylase/neopullulanase of Bacteroides thetaiotaomicron showing maltoheptaose binding [52][53][54] (http://www.cazy.org). This modules becomes important in breaking down the structure of the substrate due to the presence of two polysaccharide-binding sites [55].…”

“…Furthermore, a starch biosynthetic enzyme, the starch synthase III (SSIII) from Arabidopsis thaliana (AtSSIII) has been reported by our group to have a regulatory role in the synthesis of transient starch [33]. This enzyme contains 1025 amino acid residues and has an N-terminal transit peptide for chloroplast localization followed by three in tandem starch-binding domains (SBD D1, D2 and D3, residues 22-591), which bind to raw starch and its individual components, amylose or amylopectin [53,54,58,59]. The adsorption experiments show that the SBD123 region binds preferentially to amylose, and that the D1 domain is mainly responsible for this selective binding.…”

Polysaccharide-synthesizing Glycosyltransferases and Carbohydrate Binding Modules: the case of Starch Synthase III

“…However, our work was the first report on the existence of an SBD in a synthesizing enzyme (AtSSIII) and, the first experimental evidence of its starch binding capacity which allowed to classify these modules within the CBM53 family [35,49,50].…”

“…The D2 domain contains two polysaccharide binding sites which include amino acid residues Y394 (which is part of the binding site 1) and W366 (in the binding site 2) acting cooperatively with the D1 domain in the binding process. It was described that mutations in these residues also affect the kinetic parameters for the polysaccharide substrate of AtSSIII [50].…”