Mechanism Underlying Heat Stability of the Rice Endosperm Cytosolic ADP-Glucose Pyrophosphorylase

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

Investigation of the Interaction between the Large and Small Subunits of Potato ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. Recently the heterotetrameric structure of potato AGPase has been modeled. In the current study, we have applied the molecular mechanics generalized born surface area (MM-GBSA) method and identified critical amino acids of the potato AGPase LS and SS subunits that interact with each other during the native heterotetrameric structure formation. We have further shown the role of the LS amino acids in subunit-subunit interaction by yeast two-hybrid, bacterial complementation assay and native gel. Comparison of the computational results with the experiments has indicated that the backbone energy contribution (rather than the side chain energies) of the interface residues is more important in identifying critical residues. We have found that lateral interaction of the LS-SS is much stronger than the longitudinal one, and it is mainly mediated by hydrophobic interactions. This study will not only enhance our understanding of the interaction between the SS and the LS of AGPase, but will also enable us to engineer proteins to obtain better assembled variants of AGPase which can be used for the improvement of plant yield

ALLOSTERIC REGULATION OF THE RICE ENDOSPERM ADP-GLUCOSE PYROPHOSPHORYLASE

ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed step of starch biosynthesis in higher plants. The enzyme is allosterically activated by 3-phosphoglyceric acid (3-PGA) and inhibited by inorganic phosphate (Pi). Activity of AGPase is also controlled by redox regulation, a mechanism which tunes its activity in response to fluctuating light and sugar levels. The plant AGPases are composed of pairs of large subunits (LSs) and small subunits (SSs) which collectively comprise its heterotetrameric structure. Current evidence suggests that the SS has a dominant role in the enzyme catalysis while both the SS and the LS influence the allosteric regulatory properties of the enzyme. There are multiple isoforms of the enzyme depending on the tissue and intracellular localization. In cereal endosperm major AGPase activity is cytosolic in addition to the minor contribution from the amyloplast isoform. LS missense mutants, EM540, EM715 and EM817, of the rice endosperm cytosolic AGPase were isolated which had lower seed weights than the LS null mutants. EM540/817 and EM715 had T139I and A171V mutations in the LS, respectively. To investigate the effects of these mutations recombinant wild type and mutant AGPases as well as SS homotetrameric enzyme were expressed in Escherichia coli, purified to near homogeneity and assessed for their kinetic properties. Kinetic analysis showed that the lower seed weights of the LS missense mutants compared to the null mutants are due to the poorer allosteric inhibitory properties of the mutant enzymes than the SS homotetrameric enzyme. In a second study, activity of the wild type enzyme was demonstrated to be controlled by redox regulation through modification of the LS N-terminal. To examine the roles of cysteine residues (C12, C47 and C58) located at this region several mutant combinations were generated and kinetically characterized. The results showed that the wild type AGPase is more active and it is greater than 3-fold more responsive to 3-PGA when reduced. In addition, the LS residues, C47 and C58, are essential for proper 3-PGA response of the enzyme. Collectively, the results provide important insights about the control of starch metabolism in rice endosperm

Insights into Subunit Interactions in the Heterotetrameric Structure of Potato ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase, a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. The LS is involved in mainly allosteric regulation through its interaction with the catalytic SS. Recently the crystal structure of the SS homotetramer has been solved, but no crystal structure of the native heterotetrameric enzyme is currently available. In this study, we first modeled the three-dimensional structure of the LS to construct the heterotetrameric enzyme. Because the enzyme has a 2-fold symmetry, six different dimeric (either up-down or side-by-side) interactions were possible. Molecular dynamics simulations were carried out for each of these possible dimers. Trajectories obtained from molecular dynamics simulations of each dimer were then analyzed by the molecular mechanics/Poisson-Boltzmann surface area method to identify the most favorable dimers, one for up-down and the other for side-by-side. Computational results combined with site directed mutagenesis and yeast two hybrid experiments suggested that the most favorable heterotetramer is formed by LS-SS (side-by-side), and LS-SS (up-down). We further determined the order of assembly during the heterotetrameric structure formation. First, side-by-side LS-SS dimers form followed by the up-down tetramerization based on the relative binding free energies

ΔG_binding values of important residues in SS.

§<p>Standard error of mean. These residues are reported by Jin et al (19) in our AGPase model. Values are in kcal/mol. Note that interface residues in A and C chains are not listed since these chains are occupied with LSs in our AGPase model. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000546#s2" target="_blank">Results</a> were obtained from the free energy decomposition of LS-SS interaction (D1 and D2 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000546#pcbi-1000546-g001" target="_blank">Fig. 1b</a>).</p

Binding free energy components (kcal/mol) for each of the dimers averaged over the 200 snapshots.

<p>Values in parentheses are standard errors of the means. Explanation for the abbreviations can be found in materials and methods. ΔG_elec corresponds to sum of gas-phase electrostatic energy and polar solvation energy.</p

Free energy decomposition of hot spot residues in Dimer 2 (Values are in kcal/mol).

<p>Free energy decomposition of hot spot residues in Dimer 2 (Values are in kcal/mol).</p

Heterotetrameric assembly of mutants and wildtype potato AGPases.

<p>Western Blot analysis of various mutants of the LS and wild type SS. Top two panels belong native gels. 10 µg of total protein from crude extract were loaded on 3–13% native gradient gel and followed by western blot using anti-LS and anti-SS antibodies. Bottom two panels show western blot results from 10% SDS-PAGE using anti-LS and anti-SS antibodies.</p

Bacterial complementation assay using various mutants of the LS and the wildtype SS.

<p>Iodine vapor staining of <i>E. coli</i> AC70R1-504 (glgC⁻) containing wild-type SS potato AGPase (pML10) and mutant or wild-type LS AGPase (pML7). The plate was streaked from a single colony of each strain onto a Kornberg's 2% glucose enriched plate and incubated overnight at 37°C. From A to C plates containing various mutants of the LS and the wildtype potato AGPases.</p