Identification of a novel starch synthase III from the picoalgae Ostreococcus tauri

Abstract

Hydrosoluble glycogen is the major energy storage compound in bacteria, archaea, fungi, and animal cells. In contrast, photosynthetic eukaryotes have evolved to build a highly organized semicrystalline granule of starch. Several enzymes are involved in polysaccharide synthesis, among which glycogen or starch synthase catalyze the elongation of the α-1,4-glucan chain. Ostreococcus tauri, accumulates a single starch granule and contains three starch synthase III (SSIII) isoforms, known as OsttaSSIII-A, OsttaSSIII-B and OsttaSSIII-C. After amino acids sequence analysis we found that OsttaSSIII-C lacks starch-binding domains, being 49% identical to the catalytic region of the SSIII from Arabidopsis thaliana and 32% identical to the entire Escherichia coli glycogen synthase. The recombinant, highly purified OsttaSSIII-C exhibited preference to use as a primer branched glycans (such as rabbit muscle glycogen and amylopectin), rather than amylose. Also, the enzyme displayed a high affinity toward ADP-glucose. We found a marked conservation of the amino acids located in the catalytic site, and specifically determined the role of residues R270, K275 and E352 by site-directed mutagenesis. Results show that these residues are important for OsttaSSIII-C activity, suggesting a strong similarity between the active site of the O. tauri SSIII-C isoform and other bacterial glycogen synthases.

Starch Synthesis in Ostreococcus tauri: The Starch-Binding Domains of Starch Synthase III-B Are Essential for Catalytic Activity

“…Similar results, demonstrating highest activity with glycogen as acceptor substrate, were previously reported for ArathSSIII ( Valdez et al, 2008 ) and OsttaSSIII-C ( Barchiesi et al, 2017 ). OsttaSSIII-B did not exhibit enzymatic activity under the assayed conditions when UDPGlc was used as the donor substrate.…”

“…By contrast, the S 0.5 value for ADPGlc was 90 μM when glycogen was used as the acceptor substrate (Table 3 ). The S 0.5 value for glycogen was about 75% lower than those reported for other SSIIIs, such as OsttaSSIII-C and ArathSSIII ( S 0.5 = 0.23 and 0.26 mg/ml, respectively) ( Valdez et al, 2008 ; Wayllace et al, 2010 ; Barchiesi et al, 2017 ). On the other hand, n H values for ADPGlc and glycogen were about 1.16 and 0.98, respectively, whereas for AP and AM were 1.48 and 1.60, respectively, suggesting a cooperative kinetics for these polysaccharides.…”

“…The homogenate was loaded onto a HiTrap chelating HP column (GE Healthcare BioSciences, Uppsala, Sweden) equilibrated with binding buffer (20 mM TRIS-HCl pH 7.5, 20 mM imidazole). The column was washed with 10–15 volumes of binding buffer, and each protein was eluted using a linear gradient of binding buffer and elution buffer (20 mM TRIS-HCl pH 7.5, 20–500 mM imidazole) ( Barchiesi et al, 2017 ). The presence of OsttaSSIII-B (103.2 kDa) or OsttaSSIII-B CD (56.3 kDa) in the eluted fractions was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses.…”

“…The activity of WT and mutated OsttaSSIII-B proteins was determined following the formation of inorganic phosphate (Pi) after hydrolysis of generated ADP by alkaline phosphatase by colorimetric assays as previously reported ( Barchiesi et al, 2017 ). The reaction medium (50 μl final volume) contained 50 mM BICINE pH 8.0, 2 U of E. coli alkaline phosphatase (Sigma-Aldrich, MO, United States), 0–1 mM ADPGlc and one of the following polysaccharides: 0–2 mg/ml rabbit liver glycogen (G8876 Sigma-Aldrich), 0–3 mg/ml potato AP (10118; Fluka), 0–1 mg/ml potato AM (10130; Fluka), or 0–2 mg/ml potato starch (85649; Fluka).…”

“…Although O. tauri constitutes the smallest eukaryotic cell and possesses the smallest genome of a eukaryotic photosynthetic organism described to date, it contains more SSIII-like genes (three copies) than any other algae or plant species (e.g., Arabidopsis thaliana has only one copy) ( Deschamps et al, 2008c ). Thus, the genome of O. tauri encodes three SSIIIs (SSIII-A, SSIII-B, and SSIII-C) of which only SSIII-C has been characterized ( Barchiesi et al, 2017 ). Interestingly, the O. tauri genome lacks genes related to yeast or mammalian glycogenin.…”