Conformational Characteristics of Rice Hexokinase OsHXK7 as a Moonlighting Protein involved in Sugar Signalling and Metabolism

Hexokinase (HXK) as a moonlighting protein involves in glucose metabolism and signalling to regulate growth and development in plants. Therefore, the clarification for the structural properties of OsHXK7 (Oryza sativa Hexokinase 7) is essential to understand its role mechanism associated with the Glc signalling and metabolism. In this study, the structural characteristics of OsHXK7 (Oryza sativa Hexokinase 7) were identified. In the fluorescence spectrum, the Trp peak representing OsHXK7 binding to D-glucose (D-Glc) and 2-deoxyglucose (2-dG) showed an obvious blue shift. The distinct change in the secondary structure of OsHXK7 after binding to Glc was also detected in circular dichroism spectra. Using superimposed modelling, OsHXK7 showed a Glc-induced structural change, in which the 76th glycine, 148th serine and 256th tryptophan were contained within the pocket region. It was further shown by site-directed mutagenesis that the 76th glycine and the 256th tryptophan, but not the 148th serine, are the pivotal sites of OsHXK7 that maintain its catalytic activity and intrinsic blue shift fluorescence. These results suggest that OsHXK7 binding to Glc leads to a conformational change, that is likely essential for the function of OsHXK7 in Glc signalling and metabolism during plant growth and development.