Glucan, water dikinase (GWD) penetrates the starch granule surface and introduces C6 phosphate in the vicinity of branching points

Starch phosphorylation mediated by a-glucan, water dikinase is an integral part of starch metabolism. So far however, it is not fully understood. For getting deeper insights, several in vitro assays and intensive mass spectrometry analyses were performed. Such analyses allowed us to determine the phosphorylation position within the amylopectin in detail. Thus, unique features of the starch structure and GWD action were correlated. Therefore, recombinant potato GWD (Solanum tuberosum L.; StGWD) was used for detailed analyses of the phosphorylation pattern of various starches. Additionally, oil palm (Elaeis guineensis Jacq.; EgGWD) GWD was cloned and characterized, representing the first characterization of GWD of a monocot species. The distribution patterns of single phosphorylated glucan chains catalyzed by both GWDs were compared. The phosphorylation distribution patterns of both GWDs varied for different starches. It was proven that GWD phosphorylates different positions within the amylopectin of native starch granules. GWD enters the starch granule surface and phosphorylates the glucosyl units in the proximity of branching points to convert the highly ordered glucan chains into a less ordered state and to render them accessible for the downstream acting hydrolases. This enables deciphering the GWD actions and the related structural properties of starch granules.

Automated summary

Starch phosphorylation mediated by a-glucan, water dikinase is an important part of starch metabolism, but its mechanism is not fully understood. Through in vitro assays and mass spectrometry analyses, the phosphorylation position within amylopectin was determined, revealing the unique features of starch structure and GWD action. Different phosphorylation patterns were observed for different starches, indicating that GWD can phosphorylate different positions within native starch granules, making them more accessible for hydrolases.