Role of the N-terminal starch-binding domains on kinetic properties of starch synthase III from Arabidopsis thaliana

VALDEZ A. ,WAYLLACE, N, PARISI, G, BUSI, MV, UGALDE, R AND GOMEZ-CASATI, D

BIOCHEMISTRY

AMER CHEMICAL SOC

Año: 2008 vol. 47 p. 3026 - 3032

0006-2960

Starch synthase III (SSIII), one of the SS isoforms involved in plant starch synthesis, has beenreported to play a regulatory role in the synthesis of transient starch. SSIII from Arabidopsis thaliana contains1025 amino acid residues and has an N-terminal transit peptide for chloroplast localization which is followedby 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 fulllengthand truncated isoforms of SSIII, lacking one, two, or three SBDs, and recombinant proteins, containingthree, two, or one SBD, to investigate the role of these domains in enzyme activity. Results revealed that SSIIIuses preferentially ADPGlc, although UDPGlc can also be used as a sugar donor substrate. When ADPGlcwas used, the presence of the SBDs confers particular properties to each isoform, increasing the apparentaffinity and the Vmax for the oligosaccharide acceptor substrate. However, no substantial changes in the kineticparameters for glycogen were observed when UDPGlc was the donor substrate. Under glycogen saturatingconditions, the presence of SBDs increases progressively the apparent affinity and Vmax for ADPGlc but notfor UDPGlc. Adsorption assays showed that the N-terminal region of SSIII, containing three, two, or one SBDmodule 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 bindingcapacity and modulating the catalytic properties of SSIII.