Functional and structural characterization of the catalytic domain of the starch synthase III from Arabidopsis thaliana

Abstract

Glycogen and starch are the major energy storage compounds in most living organisms. The metabolic pathways leading to their synthesis involve the action of several enzymes, among which glycogen synthase (GS) or starch synthase (SS) catalyze the elongation of the alpha-1,4-glucan backbone. At least five SS isoforms were described in Arabidopsis thaliana; it has been reported that the isoform III (SSIII) has a regulatory function on the synthesis of transient plant starch. The catalytic C-terminal domain of A. thaliana SSIII (SSIII-CD) was cloned and expressed. SSIII-CD fully complements the production of glycogen by an Agrobacterium tumefaciens glycogen synthase null mutant, suggesting that this truncated isoform restores in vivo the novo synthesis of bacterial glycogen. In vitro studies revealed that recombinant SSIII-CD uses with more efficiency rabbit muscle glycogen than amylopectin as primer and display a high apparent affinity for ADP-Glc. Fold class assignment methods followed by homology modeling predict a high global similarity to A. tumefaciens GS showing a fully conservation of the ADP-binding residues. On the other hand, this comparison revealed important divergences of the polysaccharide binding domain between AtGS and SSIII-CD.

Characterization of Recombinant UDP- and ADP-Glucose Pyrophosphorylases and Glycogen Synthase To Elucidate Glucose-1-Phosphate Partitioning into Oligo- and Polysaccharides in Streptomyces coelicolor

“…Although the parameters for S. coelicolor GSase differ from those obtained for the E. coli homolog (44), they are very close to the parameters obtained in the characterization of the starch synthase III from Arabidopsis thaliana (14). It is well established that starch synthesis in plants and bacterial glycogen synthesis share similar properties in relation to their mechanisms and regulation steps (3,4).…”

Starch Biosynthesis in the Developing Endosperms of Grasses and Cereals

“…At least five classes of SS are found in the developing endosperm, each with distinct catalytic properties with respect to substrate preferences and α-glucan product that is formed [176]. The SSs share structural characteristics with each other, and other glycosyltransferases, e.g., glycogen synthases, including a highly conserved K-X-G-G-L motif in the C-terminal domain that is responsible for substrate binding [176][177][178][179]. Variation amongst the SS isoforms is found in the N-terminus upstream of the catalytic core, and varies in length from 2.2 kDa in granule-bound starch synthase I to approximately 135 kDa in maize SSIII [180].…”

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

“…1B). It is important to mention that the comparison between the model and GS crystal structure revealed that the global topology is almost the same, except for differences in some loops [10,48]. Thus, the catalytic domain of AtSSIII, as well as the bacterial GSs share the same GT-B fold and were classified in the GT-5 family of glycosyltransferases.…”

“…AtSSIII, with a total of 1025 amino acid residues, is a particular enzyme since it contains a putative N-terminal transit peptide followed by a 557-amino acid SSIII specific domain (SSIII-SD) and a C-terminal catalytic domain of 450 amino acids [34,48]. We previously built a 3D structural model of the catalytic domain of AtSSIII using the structure of the glycogen synthase (GS, PDB ID 1RZU) from A. tumefaciens [48] (see also Fig.…”

“…We previously built a 3D structural model of the catalytic domain of AtSSIII using the structure of the glycogen synthase (GS, PDB ID 1RZU) from A. tumefaciens [48] (see also Fig. 1B).…”