A pivoting histidine domain mediates phosphate transfer in glucan water dikinase.

As the sole naturally occurring covalent modification of starch, phosphorylation plays a critical role in regulating starch metabolism across higher plants and algae. Starch phosphorylation is catalyzed by high molar mass dikinases, such as the glucan water dikinase from Solanum tuberosum (StGWD1). This is the most extensively studied glucan dikinase, and preliminary structure prediction and comparison with other dikinases suggested a swiveling mechanism for β-phosphate transfer to the glucan substrate; however, the experimental 3D structure of StGWD1 remains largely unknown, and its structural dynamics lack experimental validation. Here, we employed biochemical characterization, AlphaFold2 modeling, X-ray crystallography, and Small-Angle X-ray Scattering (SAXS) to gain insight into the structure and mechanism of StGWD1. The protein comprises five domains, including two N-terminal carbohydrate binding domains (CBMs) followed by a central domain, whose structure was solved by X-ray crystallography in both open and closed conformations. They are followed by the domain bearing the catalytic histidine and the ATP-binding domain. Using SAXS-driven modeling, we characterized the spatial arrangement of the full-length enzyme and several truncated variants, identifying a pivoting movement of the histidine domain consistent with autophosphorylation and subsequent phosphate transfer to glucan. Our data highlight residues at the domain interfaces that may assist catalysis. Furthermore, we hypothesize that the second CBM, which remains always close to the central domain, helps maintaining the catalytic domain in proximity to the glucan chain for productive phosphate transfer.