CONICET | Buscador de Institutos y Recursos Humanos

One of the isoforms of starch-synthase (SSIII) has an N-terminal transit peptide followed by three internal starch-binding domains (SBD) and a C-terminal catalytic domain (CD). Recently, we characterized this isoform from A. thaliana and demonstrated that the SBDs have starch binding capacity and modulates the catalytic activity of the enzyme. In this work, we examined possible intramolecular interactions between the N- and C-domains which may contribute to the regulation of SSIII. We divided the enzyme in different polypeptides corresponding to the N-terminal SBDs containing three, two or one SBD (D123, D23, D3, D2 and D1) and the CD. We cloned, co-expressed, purified, perform kinetic characterization of the recombinant proteins and carried out protein-protein interaction analysis. Pull down and far western blotting assays show that D123 and D23 proteins (but not the individual domains D1, D2 or D3) physically interact with the CD. The presence of SBDs completely restores the kinetic parameters for glycogen, but only restores partially those for ADPGlc. Our data shows that the D23 region is critical for the interaction with the catalytic domain. The results presented here suggest that the N- and C- domains interaction has an important role in the modulation of A. thaliana SSIII activity.

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