CONICET | Buscador de Institutos y Recursos Humanos

Starch synthase III from Arabidopsis thaliana contains an N-terminal region, including three in-tandem starch-binding domains (SBD, named D123), followed by a C-terminal catalytic domain (CD). We had reported previously that SBDs are involved in the regulation of starch synthase III function and recently, we demonstrated protein interaction between both domains, showing that the amino acids in 316-344 and 495-535 regions in D2 and D3 domains respectively, but not the individual SBDs, are involved in interaction with catalytic domain (1,2). In this work, we used bioinformatic tools, scanning-Ala assays, pull down and kinetic activity measurements in order to determine which residues belonging to the D23 region are involved in the interaction with the CD. By SDM, we generate a battery of Ala-modified D23 proteins between aminoacids 316-344, and the interaction with the CD was evaluated using pull down assays. Results showed that S328-E330 and N338-W340 residues lost the ability to interact with the CD. When these residues were changed by Ala in the full length SSIII protein, the kinetic parameters were similar to that obtained for the D3-CD truncated enzyme. The results presented here indicate that the interaction of the N-terminal SBDs, particularly the 316-344 and 495-535 loops regions, with the catalytic domains, are important in the modulation of SSIII activity, and suggest that the S328-E330 and N338-W340 residues are involved in this process. References: 1- Valdez, H.A.; Busi, M.V.; Wayllace, N.Z.; Parisi, G.; Ugalde, R.A. and Gomez-Casati, D.F. (2008) Biochemistry 47, 3026-3032. 2- Wayllace, N.Z.; Valdez, H.A.; Ugalde, R.A.; Busi, M.V. and Gomez-Casati, D.F (2010). FEBS J 277, 428-440. Acknowledgments: PIP CONICET 112-200801-00237 y PROG07F/2 UNSAM