Enhanced Heterotetrameric Assembly of Potato ADP-Glucose Pyrophosphorylase Using Reverse Genetics

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. Computational and experimental studies have revealed that the heterotetrameric assembly of AGPase is thermodynamically weak. Modeling studies followed by the mutagenesis of the LS of the potato AGPase identified a heterotetramer-deficient mutant, LS(R88A). To enhance heterotetrameric assembly, LS(R88A) cDNA was subjected to error-prone PCR, and second-site revertants were identified according to their ability to restore glycogen accumulation, as assessed with iodine staining. Selected mutations were introduced into the wild-type (WT) LS and co-expressed with the WT SS in Escherichia coli glgC(-). The biochemical characterization of revertants revealed that LS(I90V)SS(WT), LS(Y378C)SS(WT) and LS(D410G)SS(WT) mutants displayed enhanced heterotetrameric assembly with the WT SS. Among these mutants, LS(Y378C)SS(WT) AGPase displayed increased heat stability compared with the WT enzyme. Kinetic characterization of the mutants indicated that the LS(I90V)SS(WT) and LS(Y378C)SS(WT) AGPases have comparable allosteric and kinetic properties. However, the LS(D410G)SS(WT) mutant exhibited altered allosteric properties of being less responsive and more sensitive to 3-phosphoglyceric acid activation and inorganic phosphate inhibition. This study not only enhances our understanding of the interaction between the SS and the LS of AGPase but also enables protein engineering to obtain enhanced assembled heat-stable variants of AGPase, which can be used for the improvement of plant yields.

Analysis of the rice ADPglucose transporter (OsBT1) indicates the presence of regulatory processes in the amyloplast stroma that control ADPglucose flux into starch

“…ADPglc content was then estimated for the purified amyloplast fraction and total seed extract by performing the reverse AGPase reaction coupled with phosphoglucomutase and Glc-6-P dehydrogenase reactions as described previously (Seferoglu et al, 2014). The reaction was initiated by the addition of excess amounts of near homogenously pure potato AGPase and performed at 37°C for 20 min in reaction buffer containing 100 mM HEPES, pH 7, 5 mM dithiothreitol, 5 mM 3-phosphoglyceric acid, 10 mM MgCl 2 , 0.6 mM NAD + , 0.4 mg mL 21 bovine serum albumin, 0.5 units of Glc-6-P dehydrogenase, and 0.5 units of phosphoglucomutase.…”

Glu-370 in the Large Subunit Influences the Substrate Binding, Allosteric, and Heat Stability Properties of Potato ADP-glucose Pyrophosphorylase

“…The heterotetrameric assembly properties of the LS E370G mutant AGPase with wild type SS were investigated using 3-13% gradient native polyacrylamide gels followed by Western blot using potato anti-LS and anti-SS antibodies as previously described [27].…”

“…Modeling studies followed by mutagenesis of the potato AGPase identified a hotspot mutant AGPase for the interaction of head to tail LS-SS dimers, where an Arg residue at position 88 was replaced by Ala in the LS (LS R88A ) [30,34]. In our previous study we employed an error prone-PCR random mutagenesis approach using the LS A88 AGPase cDNA as template to select second-site LS suppressor that could reverse the iodine staining deficiency of the E.coli glgCcontaining wild type SS AGPase cDNA [27].…”

“…Stained colonies were picked up and plasmids were isolated. To identify mutations each mutant were subjected to the sequencing as described in [27]. To see the effect of the second site revertants independently in AGPase function, plasmids containing mutant LS cDNAs were then purified and subjected to site-directed mutagenesis by PCR to convert the primary mutation Ala88 back to the corresponding wild type arginine residue.…”

“…Thermal stability of the enzyme has been shown to not only depend on the robustness of the LS-SS interactions but also on the binding of the effector molecules to the allosteric sites [23,26,27].…”