Crystal structures of rice hexokinase 6 with a series of substrates shed light on its enzymatic mechanism.

Hexokinases (HXKs) have determined to be multifaceted proteins, and they are the only ones able to phosphorylate glucose in plants. However, the binding mode for ATP to plant HXKs remains unclear. Here, we report the crystal structures of rice hexokinase 6 (OsHXK6) in four different forms: (i) apo-form, (ii) binary complex with D-Glc, (iii) quaternary complex with ADP, PO and Mg, and (iv) pentanary complex with D-Glc, ADP, PO, and Mg. The apo form is in the open state conformation, and the three others are in the closed state, indicating that glucose and ADP-PO binding induces a large conformational change by domain rearrangement. The quaternary complex is a novel intermediate during the catalytic reaction we trapped for the first time, which provides a new evidence for the enzymatic mechanism of HXKs. In addition, the latter two complexes reveal the binding mode for ADP-PO to plant HXKs, which provide the structural explanation for the dual-function of OsHXK6. In addition, we identified that residues Gly112, Thr261, Gly262, and Gly450 are essential to the binding between ADP-PO and OsHXK6 by a series of single mutations and enzymatic assays. Our study provide structural basis for the other functional studies of OsHXK6 in rice.