Abstract
Starch is the major energy storage carbohydrate in photosynthetic eukaryotes. Several enzymes are involved in building highly organized semi-crystalline starch granules, including starch-synthase III (SSIII), which is widely conserved in photosynthetic organisms. This enzyme catalyzes the extension of the α-1,4 glucan chain and plays a regulatory role in the synthesis of starch. Interestingly, unlike most plants, the unicellular green alga Ostreococcus tauri has three SSIII isoforms. In the present study, we describe the structure and function of OsttaSSIII-B, which has a similar modular organization to SSIII in higher plants, comprising three putative starch-binding domains (SBDs) at the N-terminal region and a C-terminal catalytic domain (CD). Purified recombinant OsttaSSIII-B displayed a high affinity toward branched polysaccharides such as glycogen and amylopectin, and to ADP-glucose. Lower catalytic activity was detected for the CD lacking the associated SBDs, suggesting that they are necessary for enzyme function. Moreover, analysis of enzyme kinetic and polysaccharide-binding parameters of site-directed mutants with modified conserved aromatic amino acid residues W122, Y124, F138, Y147, W279, and W304, belonging to the SBDs, revealed their importance for polysaccharide binding and SS activity. Our results suggest that OT_ostta13g01200 encodes a functional SSIII comprising three SBD domains that are critical for enzyme function.
Highlights
Starch plays a key role in the life cycle of plants as the primary form of carbohydrate storage for chemical energy
Our results indicate that the domain organization of OsttaSSIII-B is most similar to the synthase III (SSIII) from higher plants than the other two OsttaSSIII isoforms
The starch binding sites described for ArathSSIII D2 are highly conserved in OsttaSSIII-B starch-binding domains (SBDs), residues G335, W340, and Y394 (ArathSSIII numbering) belonging to binding site 1 (Valdez et al, 2011; Barchiesi et al, 2015)
Summary
Starch plays a key role in the life cycle of plants as the primary form of carbohydrate storage for chemical energy. It has acquired immense value as a vital source of energy and as a precursor of new biodegradable materials (Ball and Morell, 2003; Sticklen, 2006). Starch accumulates as semicrystalline granules with a complex structure and organization composed of two polymers, amylose (AM) and amylopectin (AP). In both polymers, glucose is connected by α-1,4 glycosidic bonds, forming linear chains, and α-1,6 bonds in the so-called branch points. AP has a larger number of branches (5%) and is the dominant component, accounting for 65–85% of granules (Nakamura, 2002; Zeeman et al, 2010; Pfister and Zeeman, 2016)
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