Starch synthase III (SSIII), one of the SS isoforms involved in plant starch synthesis, 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 which is followed by three repeated starch-binding domains (SBDs; SSIII residues 22-591) and a C-terminal catalytic domain (residues 592-1025) similar to bacterial glycogen synthase. In this work, we constructed recombinant full-length and truncated isoforms of SSIII, lacking one, two, or three SBDs, and recombinant proteins, containing three, two, or one SBD, to investigate the role of these domains in enzyme activity. Results revealed that SSIII uses preferentially ADPGlc, although UDPGlc can also be used as a sugar donor substrate. When ADPGlc was used, the presence of the SBDs confers particular properties to each isoform, increasing the apparent affinity and the V max for the oligosaccharide acceptor substrate. However, no substantial changes in the kinetic parameters for glycogen were observed when UDPGlc was the donor substrate. Under glycogen saturating conditions, the presence of SBDs increases progressively the apparent affinity and V max for ADPGlc but not for UDPGlc. Adsorption assays showed that the N-terminal region of SSIII, containing three, two, or one SBD module have increased capacity to bind starch depending on the number of SBD modules, with the D23 protein (containing the second and third SBD module) being the one that makes the greatest contribution to binding. The results presented here suggest that the N-terminal SBDs have a regulatory role, showing a starch binding capacity and modulating the catalytic properties of SSIII.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.