Jun 4, 2014

Enhanced heterotetrameric assembly of potato ADP-glucose pyrophosphorylase using reverse genetics

Plant & Cell Physiology
Ayse Bengisu SeferogluI Halil Kavakli

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 al...Continue Reading

  • References35
  • Citations3

Mentioned in this Paper

Allosteric Site
Iodine, Homeopathic preparation
Mutagenesis, Site-Directed
Solanum tuberosum
Iodine
Phosphate Measurement
Protein Multimerization
Metabolic Inhibition
Mutant
Glucose-1-phosphate adenylyltransferase

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