title: Role of the N-terminal starch-binding domains in the kinetic properties of starch synthase III from Arabidopsis thaliana.
abstract: 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.
authors.affiliation: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH), CONICET/UNSAM, Camino Circunvalación Km 6, 7130 Chascomús, Argentina.
authors.forename: Hugo A
authors.initials: HA
authors.lastname: Valdez
authors.forename: Maria V
authors.initials: MV
authors.lastname: Busi
authors.forename: Nahuel Z
authors.initials: NZ
authors.lastname: Wayllace
authors.forename: Gustavo
authors.initials: G
authors.lastname: Parisi
authors.forename: Rodolfo A
authors.initials: RA
authors.lastname: Ugalde
authors.forename: Diego F
authors.initials: DF
authors.lastname: Gomez-Casati
mesh_terms: D000245:Adenosine Diphosphate Glucose / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006003:Glycogen / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013213:Starch / Q000378:metabolism*; D013214:Starch Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D000245:Adenosine Diphosphate Glucose; D013213:Starch; D006003:Glycogen; D013214:Starch Synthase; D014532:Uridine Diphosphate Glucose