The starch-binding capacity of the noncatalytic SBD2 region and the interaction between the N- and C-terminal domains are involved in the modulation of the activity of starch synthase III from Arabidopsis thaliana.
Starch synthase III from Arabidopsis thaliana contains an N-terminal region, including three in-tandem starch-binding domains, followed by a C-terminal catalytic domain. We have reported previously that starch-binding domains may be involved in the regulation of starch synthase III function. In this work, we analyzed the existence of protein interactions between both domains using pull-down assays, far western blotting and co-expression of the full and truncated starch-binding domains with the catalytic domain. Pull-down assays and co-purification analysis showed that the D(316-344) and D(495-535) regions in the D2 and D3 domains, respectively, but not the individual starch-binding domains, are involved in the interaction with the catalytic domain. We also determined that the residues W366 and Y394 in the D2 domain are important in starch binding. Moreover, the co-purified catalytic domain plus site-directed mutants of the D123 protein lacking these aromatic residues showed that W366 was key to the apparent affinity for the polysaccharide substrate of starch synthase III, whereas either of these amino acid residues altered ADP-glucose kinetics. In addition, the analysis of full-length and truncated proteins showed an almost complete restoration of the apparent affinity for the substrates and V(max) of starch synthase III. The results presented here suggest that the interaction of the N-terminal starch-binding domains, particularly the D(316-344) and D(495-535) regions, with the catalytic domains, as well as the full integrity of the starch-binding capacity of the D2 domain, are involved in the modulation of starch synthase III activity.
Wayllace
Nahuel Z
NZ
Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, Argentina.
Valdez
Hugo A
HA
Ugalde
Rodolfo A
RA
Busi
Maria V
MV
Gomez-Casati
Diego F
DF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; D017931:DNA Primers; D018744:DNA, Plant; D011994:Recombinant Proteins; D013213:Starch; D005964:Glucosyltransferases; C504530:starch synthase III, Arabidopsis