Identification of aminoacid residues in SBD involved in the modulation of the activity of starch synthase III

WAYLLACE, N. Z.; VALDEZ, H.; BUSI, M. V.; DIEGO FABIAN GOMEZ CASATI

Potrero de los Funes

Congreso; XLVII Reunion SAIB; 2011

Soc Arg Bioquimica y Biologia Molecular

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 previously reported 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. We performed bioinformatic analysis, alanine-scanning assays, pull down and enzyme activity measurements in order to determine which residues of the D23 region are involved in the interaction with the CD. We generated different Ala-modified D23 proteins by SDM between aminoacids 316-344, and evaluated the interaction with the CD 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.