Abstract
ADP-glucose pyrophosphorylase (AGPase) is a key allosteric enzyme in plant starch biosynthesis. Plant AGPase is a heterotetrameric enzyme that consists of large (LS) and small subunits (SS), which are encoded by two different genes. In this study, we showed that the conversion of Glu to Gly at position 370 in the LS of AGPase alters the heterotetrameric stability along with the binding properties of substrate and effectors of the enzyme. Kinetic analyses revealed that the affinity of the LSE370GSSWT AGPase for glucose-1-phosphate is 3-fold less than for wild type (WT) AGPase. Additionally, the LSE370GSSWT AGPase requires 3-fold more 3-phosphogyceric acid to be activated. Finally, the LSE370GSSWTAGPase is less heat stable compared with the WT AGPase. Computational analysis of the mutant Gly-370 in the 3D modeled LS AGPase showed that this residue changes charge distribution of the surface and thus affect stability of the LS AGPase and overall heat stability of the heterotetrameric AGPase. In summary, our results show that LSE370 intricately modulate the heat stability and enzymatic activity of potato the AGPase.
Highlights
Starch is a staple in the diet of much of the world’s population and is widely used in different industries as a raw material [1]
The first committed step of starch biosynthesis in plants is catalyzed by ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27)
The LS cDNAs containing only the secondary mutations were co-expressed with wild type SS in E.coli glgC- and their activity were assessed by iodine staining
Summary
Starch is a staple in the diet of much of the world’s population and is widely used in different industries as a raw material [1]. The modeling studies enable us to identify critical amino acid residues at the interface of the AGPase that are important for the LS-SS interaction and allosteric regulations [30-‐34]. There are several studies showed the importance of heterotetrameric assemblies in enzyme functions [13, 35-‐37], amino acids that contribute structural stability of heterotetrameric AGPase is not well defined. We demonstrated that an amino acid (LSE370), close to the LS interface, is important for maintaining the heat stability of the heterotetrameric structure of the AGPase. Our data demonstrate Glu-370 is critical for the structural stability of the LS subunit itself and the overall heterotetrameric AGPase, and influences the allosteric, substrate binding and heat stability properties of the AGPase
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