Carrillo Julieta B, Gomez-Casati Diego F, Martín Mariana, Busi Maria V
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina.
PLoS One. 2018 Jan 23;13(1):e0191621. doi: 10.1371/journal.pone.0191621. eCollection 2018.
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.
莱茵衣藻是目前已知的最小的自由生活（非共生）真核生物，是绿藻纲绿藻科的单细胞绿藻。它具有非常简单的细胞结构，呈现出独特的淀粉颗粒和叶绿体。然而，其淀粉代谢表现出与高等植物相当的复杂性，每个代谢反应都有多种酶形式。葡聚糖磷酸酶是一类在动植物中功能保守的酶，分别对植物和哺乳动物中正常的淀粉或糖原降解至关重要。尽管莱茵衣藻微藻在进化中很重要，但关于参与淀粉可逆磷酸化的酶尚无相关信息。在此，我们报道了莱茵衣藻双特异性磷酸酶（OsttaDSP）基因的分子克隆和异源表达，该基因与拟南芥LSF2同源。重组酶经纯化达到电泳纯，以准确表征其寡聚和动力学性质。OsttaDSP是一种54.5 kDa的同型二聚体，可结合支链淀粉并使其去磷酸化。此外，我们还确定残基C162参与催化，可能还参与酶的结构稳定性。我们的结果有助于更好地理解葡聚糖磷酸酶在绿藻淀粉代谢中的作用。
