Abstract
The ADP-glucose pyrophosphorylase from wheat endosperm controls starch synthesis in seeds and has unique regulatory properties compared to others from this family. It comprises two types of subunits, but despite its importance little is known about their roles. Here, we synthesized de novo the wheat endosperm ADP-glucose pyrophosphorylase small (S) and large (L) subunit genes, heterologously expressed them in Escherichia coli, and kinetically characterized the recombinant proteins. To understand their distinct roles, we co-expressed them with well characterized subunits from the potato tuber enzyme to obtain hybrids with one S subunit from one source and an L subunit from the other. After kinetic analyses of these hybrids, we concluded that the unusual insensitivity to activation of the wheat endosperm enzyme is caused by a pre-activation of the L subunit. In addition, the heat stability and sensitivity to phosphate are given by the S subunit.
Highlights
One-fifth of all calories consumed in the world come from wheat
To deepen the study of the regulatory properties of wheat endosperm ADP-Glc ADP-glucose pyrophosphorylase (PPase), it was necessary to obtain a recombinant version of the enzyme
We artificially synthesized the respective genes coding for the S and L subunits with the codon usage for E. coli to maximize the heterologous expression in this bacterium
Summary
One-fifth of all calories consumed in the world come from wheat. In order to meet the caloric needs of our growing population, it is estimated that the yield of this crop must substantially increase in the near future (Altman, 1999). Crop yield is influenced by starch synthesis (Smidansky et al, 2003; Tuncel and Okita, 2013; Zi et al, 2018), which in higher plants and unicellular algae is controlled to a large extent by ADP-glucose (ADP-Glc) pyrophosphorylase (EC: 2.7.7.27; ADP-Glc PPase) This regulatory enzyme catalyzes the conversion of ATP and glucose-1-phosphate (Glc1P) to form ADP-Glc and pyrophosphate (PPi) (Iglesias and Preiss, 1992; Ballicora et al, 2003, 2004). The wheat endosperm enzyme, like other plant ADP-Glc PPases, comprises two distinct homologous subunits, small (S) and large (L), which form an S2L2 heterotetramer This endosperm form presents unique regulatory properties compared with enzymes from other sources. Despite the relevance of this particular enzyme, its impact on the agricultural importance of wheat, and its peculiar properties, little is known about their relationship to structure
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