Multiple Starch Synthase III Isoforms in the Smallest Free Living Eukaryote Ostreococcus tauri

J. BARCHIESI; M.A. BALLICORA; N. HEDIN; A.A. IGLESIAS; D.F. GOMEZ CASATI; M.V. BUSI

Rosario

Congreso; 50th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2014

SAIB

Starch synthase III from Arabidopsis thaliana contains an N terminal region, including three in tandem starch binding domains (SBD), followed by a C terminal catalytic domain. These SBDs are essential for starch binding and are involved in the regulation of catalytic activity. In Ostreococcus tauri, a unicellular green alga, there are three SSIII isoforms. Using in silico characterization techniques, we have shown that these three isoforms contain two,three and no N terminal SBD. In this work, we characterized the starch synthase activity of the three isoforms, and the involvement of SBDs in this function. For this purpose, the full length enzymes as well as truncated isoforms lacking SBDs was cloned, expressed, and purified from Escherichia coli cells, and their kinetic parameters were determined using a new discontinuous colorimetric assay. Our results indicate that the O. tauri SSIII SBDs present polysaccharide binding ability and modulate the catalytic properties of SSIII. In addition, using co sedimentation binding assays, we observed that these domains displayed some promiscuity in binding to different polysaccharide substrates. These results not only reveal significant information concerning functional aspects of SSIII and SBD domains, butare also crucial to understand the evolutionary conservation of multiple SSIII isoforms in this small picoalga.