Characterization of a novel Ostreococcus tauri phosphoglucan phosphatase likely involved in starch metabolism.

CARRILLO J.B.; MARTIN, M.; DIEGO FABIAN GOMEZ CASATI; BUSI, M. V.

Mar del Plata

Congreso; LI Reunión de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2015

SAIB

Phosphoglucan phosphatases (PP)remove phosphates from complex carbohydrates. In plants, these enzymes arevital components in the remobilization of leaf starch at night. To understand the evolution of catalysis and regulation ofthese 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 similarityto 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.