Identification and characterization of ChlreSEX4, a novel glucan phosphatase from Chlamydomonas reinhardtii green alga.

Chlamydomonas reinhardtii is the best known unicellular green alga model which has long been used to investigate all kinds of cellular processes, including starch metabolism. Here we identified and characterized a novel enzyme, ChlreSEX4, orthologous to glucan phosphatase SEX4 from Arabidopsis thaliana, that is capable of binding and dephosphorylating amylopectin in vitro. We also reported that cysteine 224 and tryptophan 305 residues are critical for enzyme catalysis and substrate binding. Furthermore, we verified that ChlreSEX4 gene is expressed in vivo and that glucan phosphatase activity is measurable in Chlamydomonas protein extracts. In view of the results presented, we suggest ChlreSEX4 as a functional phosphoglucan phosphatase from C. reinhardtii. Our data obtained so far contribute to understanding the phosphoglucan phosphatases evolutionary process in the green lineage and their role in starch reversible phosphorylation. In addition, this allows to position Chlamydomonas as a potential tool to obtain starches with different degrees of phosphorylation for industrial or biotechnological purposes.

Development of fast and simple chromogenic methods for glucan phosphatases in-gel activity assays.

Glucan phosphatases are essential for normal starch degradation in plants and glycogen metabolism in mammals. Here we develop two chromogenic methods for the detection of glucan phosphatase activity in situ after non denaturing poliacrylamide gel electrophoresis; one method uses pNPP and the second one applies BCIP/NBT. The assays are sensitive, fast, simple, reliable and cost-effective preventing the use of radioactive or fluorogenic compounds. Taking advantage of an efficient separation method combined with the reported assays it is possible to obtain information about oligomeric state of the active enzymes as well as to simultaneously detect glucan substrate binding and phosphatase activity.

Identification and analysis of OsttaDSP, a phosphoglucan phosphatase from Ostreococcus tauri.

Ostreococcus tauri, the smallest free-living (non-symbiotic) eukaryote yet described, is a unicellular green alga of the Prasinophyceae family. It has a very simple cellular organization and presents a unique starch granule and chloroplast. However, its starch metabolism exhibits a complexity comparable to higher plants, with multiple enzyme forms for each metabolic reaction. Glucan phosphatases, a family of enzymes functionally conserved in animals and plants, are essential for normal starch or glycogen degradation in plants and mammals, respectively. Despite the importance of O. tauri microalgae in evolution, there is no information available concerning the enzymes involved in reversible phosphorylation of starch. Here, we report the molecular cloning and heterologous expression of the gene coding for a dual specific phosphatase from O. tauri (OsttaDSP), homologous to Arabidopsis thaliana LSF2. The recombinant enzyme was purified to electrophoretic homogeneity to characterize its oligomeric and kinetic properties accurately. OsttaDSP is a homodimer of 54.5 kDa that binds and dephosphorylates amylopectin. Also, we also determined that residue C162 is involved in catalysis and possibly also in structural stability of the enzyme. Our results could contribute to better understand the role of glucan phosphatases in the metabolism of starch in green algae.