Abstract
Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. ADP-glucose pyrophosphorylase (AGPase) is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. To increase the heat tolerance of the rice enzyme, we engineered two dominant AGPase subunits expressed in developing endosperm, the large (L2) and small (S2b) subunits of the cytosol-specific AGPase. Bacterial expression of the rice S2b with the rice L2, potato tuber LS (pLS), or with the mosaic rice-potato large subunits, L2-pLS and pLS-L2, produced heat-sensitive recombinant enzymes, which retained less than 10% of their enzyme activities after 5 min incubation at 55°C. However, assembly of the rice L2 with the potato tuber SS (pSS) showed significantly increased heat stability comparable to the heat-stable potato pLS/pSS. The S2b assembled with the mosaic L2-pLS subunit showed 3-fold higher sensitivity to 3-PGA than L2/S2b, whereas the counterpart mosaic pLS-L2/S2b showed 225-fold lower sensitivity. Introduction of a QTC motif into S2b created an N-terminal disulfide linkage that was cleaved by dithiothreitol reduction. The QTC enzyme showed moderate heat stability but was not as stable as the potato AGPase. While the QTC AGPase exhibited approximately fourfold increase in 3-PGA sensitivity, its substrate affinities were largely unchanged. Random mutagenesis of S2bQTC produced six mutant lines with elevated production of glycogen in bacteria. All six lines contained a L379F substitution, which conferred enhanced glycogen production in bacteria and increased heat stability. Modeled structure of this mutant enzyme revealed that this highly conserved leucine residue is located in the enzyme’s regulatory pocket that provides interaction sites for activators and inhibitors. Our molecular dynamic simulation analysis suggests that introduction of the QTC motif and the L379F mutation improves enzyme heat stability by stabilizing their backbone structures possibly due to the increased number of H-bonds between the small subunits and increased intermolecular interactions between the two SSs and two LSs at elevated temperature.
Highlights
Starch is a major component in many plant seed endosperms, accounting for 56–74% of the available carbohydrates in the grain of most food crops including cereals (Koehler and Wieser, 2013)
When co-expressed in E. coli, the rice ADP-Glucose pyrophosphorylase (AGPase) L2/S2b was capable of producing glycogen (Figure 1A), at a much lower extent than that evident for the potato potato tuber large subunits (LS) (pLS)/potato tuber SS (pSS) (Figure 1B)
Mosaic rice-potato LS enzymes (Supplementary Figure S1) were tested. These were pLS-L2 where the potato LS N-terminal region was fused to the rice L2 β-helix domain and L2-pLS where the rice L2 N-terminal region was fused to potato LS β-helix domain
Summary
Starch is a major component in many plant seed endosperms, accounting for 56–74% of the available carbohydrates in the grain of most food crops including cereals (Koehler and Wieser, 2013). In order to test whether the introduction of disulfide bond between the two S2b subunits increases the heat stability of the rice AGPase as demonstrated for the maize AGPase (Linebarger et al, 2005), we replaced the N-terminal NKN peptide of S2b with QTC to generate QTCL, a peptide conserved in heat stable AGPases. The hybrid pLS/S2b showed very low heat stability much like the rice AGPase This result indicates that the small subunit of the AGPase enzyme plays a more important role for enzyme’s heat stability than the large subunit. The QTC mutant retained more than 22 and 16% of enzyme activity after 5 min and 10 min incubation at 55◦C, respectively, indicating the rice enzyme acquired partial heat stability by the peptide replacement
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