Abstract
ADP-glucose pyrophosphorylase (AGPase) is an important enzyme in starch synthesis and previous studies showed that the heat lability of this enzyme is a determinant to starch synthesis in the maize endosperm and, in turn, seed yield. Here, amino acids in the AGPase endosperm small subunit with high B-factors were mutagenized and individual changes enhancing heat stability and/or kinetic parameters in an Escherichia coli expression system were chosen. Individual mutations were combined and analyzed. One triple mutant, here termed Bt2-BF, was chosen for further study. Combinations of this heat stable, 3-PGA-independent small subunit variant with large subunits also heat stable yielded complex patterns of heat stability and kinetic and allosteric properties. Interestingly, two of the three changes reside in a protein motif found only in AGPases that exhibit high sensitivity to 3-PGA. While not the 3-PGA binding site, amino acid substitutions in this region significantly alter 3-PGA activation kinetics.
Highlights
The heterotetrameric enzyme, ADP-glucose pyrophosphorylase (AGPase; EC 2.7.7.27), catalyzes the formation of PPi and ADP-glucose (ADP-Glc) from ATP and glucose-1-phosphate (G-1-P)
Saturation mutagenesis was performed on the Escherichia coli expression plasmid for the maize endosperm AGPase small subunit, pMONcBt2, at the nine amino acid sites with high Bfactors identified previously (Boehlein et al, 2015) (Table 1)
Transformants were grown in the presence of glucose and the accumulated glycogen was revealed by exposing bacterial colonies to iodine vapor
Summary
The heterotetrameric enzyme, ADP-glucose pyrophosphorylase (AGPase; EC 2.7.7.27), catalyzes the formation of PPi and ADP-glucose (ADP-Glc) from ATP and glucose-1-phosphate (G-1-P). The glucose of ADP-Glc is used for starch synthesis. AGPase is an important control point in starch synthesis and checkpoints in the control of AGPase include allostery, transcription and posttranslational modification (reviewed in Ballicora et al, 2003; Hannah, 2007; Hannah and Green, 2008; Hannah and James, 2008; Preiss, 2009). Heat lability is a important parameter for AGPases expressed in the endosperm (Preiss et al, 1971) since past studies point to grain loss from hot weather being linked to the heat lability of this enzyme (Greene and Hannah, 1998; Hannah et al, 2012, 2017). While heat lability of AGPase might have been evolutionarily desirable for the perpetuation of the species through the ages (Greene and Hannah, 1998), it is not ideal for maize kernel yield.
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