| Starch has wide applications in nutrition and industry. The nutritional value of the main cereal crops originates from the starch content of seeds. The allosteric enzyme, ADP-glucose pyrophosphorylase (AGPase) catalyses the synthesis of ADP-glucose, a rate limiting step in starch synthesis. Modification of the regulatory properties of AGPase results in increased starch yields in several main crops. As a consequence, the objective of these studies is to identify regions in the subunits important in regulation and activity to identify sites modifiable for future agronomic gain. Plant AGPases are heterotetramers activated by 3PGA and inhibited by Pi. We exploit an Escherichia coli expression system and mosaic AGPases composed of potato tuber and maize endosperm subunit fragments to unravel this question. While potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes. Since potato tuber and maize endosperm AGPases exhibit different regulatory properties, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation. First we identified a region of 24 amino acids in the potato tuber small subunit important for activity with the potato tuber large subunit. Second, we determined that sites influencing regulatory differences map to both subunits. Extent of 3PGA activation and 3PGA affinity were found to be separate entities which mapped to different regions in both subunits. Furthermore, two mosaic AGPases displayed sigmoidal curves of 3PGA saturation. This suggests that subunit interactions are altered. Third, we have identified a mosaic insensitive to inhibition, which represents a good candidate for potential maize yield improvements. |
