title: 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.
authors.affiliation: Department of Chemical and Biological Engineering, Koc University, Rumeli Feneri Yolu, 34450 Sariyer, Turkey.
authors.forename: A Bengisu
authors.initials: AB
authors.lastname: Seferoglu
authors.affiliation: Department of Chemical and Biological Engineering, Koc University, Rumeli Feneri Yolu, 34450 Sariyer, Turkey.
authors.forename: Kaan
authors.initials: K
authors.lastname: Koper
authors.affiliation: Department of Molecular Biology and Genetics, Koc University, Rumeli Feneri Yolu, 34450 Sariyer, Turkey.
authors.forename: F Betul
authors.initials: FB
authors.lastname: Can
authors.affiliation: Istanbul University, Department of Biology, 34134 Suleymaniye, Istanbul, Turkey.
authors.forename: Gul
authors.initials: G
authors.lastname: Cevahir
authors.affiliation: Department of Chemical and Biological Engineering, Koc University, Rumeli Feneri Yolu, 34450 Sariyer, TurkeyDepartment of Molecular Biology and Genetics, Koc University, Rumeli Feneri Yolu, 34450 Sariyer, Turkey hkavakli@ku.edu.tr.
authors.forename: I Halil
authors.initials: IH
authors.lastname: Kavakli
mesh_terms: D000495:Allosteric Site; D000595:Amino Acid Sequence; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D059386:Reverse Genetics; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics
chemical_list: D051599:Glucose-1-Phosphate Adenylyltransferase
keywords: ADP-glucose pyrophosphorylase; Allosteric regulation; In vitro Mutagenesis; Protein assembly