Characterization of a novel Ostreococcus tauri Phosphoglucan Phosphatase possibly involved in starch metabolism

CARRILLO, J.; MARTIN, M; GOMEZ-CASATI, D.F.; BUSI, M.V.

MAR DEL PLATA

Congreso; 51 Annual Meeting Argentine Society for Biochemistry and Molecular Biology / LI Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular SAIB; 2015

SAIB

Phosphoglucan phosphatases (PP) remove phosphates from complex carbohydrates. In plants, these enzymes are vital components in the remobilization of leaf starch at night. To understand the evolution of catalysis and regulation of these enzymes we decided to investigate their existence in the tiniest eukaryotic green algae Ostreococcus tauri. O. tauri transcriptome BLAST searches revealed one locus encoding a protein with high sequence similarity to plant PP. Through an exhaustive in silico analysis, we concluded that the putative protein could be a chloroplastic enzyme which shares several structural features with its plant counterparts. To further characterize this finding the cDNA was cloned to express and purify the recombinant protein in E. coli cells. In vitro, we verified phosphatase activity of the recombinant enzyme against the non-specific substrate p-Nitrophenyl Phosphate as well as its ability to bind polysaccharides. Besides, by native PAGE we could determine that this enzyme could act as a dimer. Finally, glucan-phosphatase activity, on its real substrate, will be assayed in order to strength the idea of its role in starch metabolism.