Improved A. tumefaciens Glycogen Synthase by Addition of an SBD from Arabidopsis thaliana Starch Sinthase III

MARTIN M.; WAYLLACE NZ; VALDEZ HA; GOMEZ-CASATI DF; BUSI MV

Mendoza

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2012

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Starch synthase III (SSIII) from is a SS Arabidopsis thaliana isoform with a particular primary structure organization; the C- terminal domain, highly conserved in other SS isoforms, is preceded by a unique specific domain (SSIII-SD) which contains three in tandem starch binding domains (SBDs, named D1, D2 and D3) characteristic of degrading enzymes. These N-terminal SBDs have a probed regulatory role in SSIII activity, showing starch binding ability and modulating the catalytic properties of the enzyme. To further investigate the functional role of A. thaliana SSIII-SD, three His tagged chimeric proteins were constructed combining the SBDs from with the glycogen synthase (GS) from Agrobacterium tumefaciens, which lacks SBDs. Recombinant chimeric proteins were expressed and purified to homogeneity from Escherichia coli in order to kinetically characterize them. Furthermore, chimeric enzymes, capability to restore in vivo glycogen biosynthesis in E. coli cells was also tested. The results obtained showed that the D3-GS enzyme showed increased capacity of glycogen synthesis in vivo whith minor changes in their in vitro kinetic parameters.