Publications

• The rice endosperm ADP-glucose pyrophosphorylase large subunit is essential for optimal catalysis and allosteric regulation of the heterotetrameric enzyme.

  Tuncel A., Kawaguchi J., Ihara Y., Matsusaka H., Nishi A., Nakamura T., Kuhara S., Hirakawa H., Nakamura Y., Cakir B., Nagamine A., Okita T.W., Hwang S.K., Satoh H.

  Although an alternative pathway has been suggested, the prevailing view is that starch synthesis in cereal endosperm is controlled by the activity of the cytosolic isoform of ADPglucose pyrophosphorylase (AGPase). In rice, the cytosolic AGPase isoform is encoded by the OsAGPS2b and OsAGPL2 genes, ... >> More

  Although an alternative pathway has been suggested, the prevailing view is that starch synthesis in cereal endosperm is controlled by the activity of the cytosolic isoform of ADPglucose pyrophosphorylase (AGPase). In rice, the cytosolic AGPase isoform is encoded by the OsAGPS2b and OsAGPL2 genes, which code for the small (S2b) and large (L2) subunits of the heterotetrameric enzyme, respectively. In this study, we isolated several allelic missense and nonsense OsAGPL2 mutants by N-methyl-N-nitrosourea (MNU) treatment of fertilized egg cells and by TILLING (Targeting Induced Local Lesions in Genomes). Interestingly, seeds from three of the missense mutants (two containing T139I and A171V) were severely shriveled and had seed weight and starch content comparable with the shriveled seeds from OsAGPL2 null mutants. Results from kinetic analysis of the purified recombinant enzymes revealed that the catalytic and allosteric regulatory properties of these mutant enzymes were significantly impaired. The missense heterotetramer enzymes and the S2b homotetramer had lower specific (catalytic) activities and affinities for the activator 3-phosphoglycerate (3-PGA). The missense heterotetramer enzymes showed more sensitivity to inhibition by the inhibitor inorganic phosphate (Pi) than the wild-type AGPase, while the S2b homotetramer was profoundly tolerant to Pi inhibition. Thus, our results provide definitive evidence that starch biosynthesis during rice endosperm development is controlled predominantly by the catalytic activity of the cytoplasmic AGPase and its allosteric regulation by the effectors. Moreover, our results show that the L2 subunit is essential for both catalysis and allosteric regulatory properties of the heterotetramer enzyme. << Less

  Plant Cell Physiol. 55:1169-1183(2014) [PubMed] [EuropePMC]

• Identification of the ADP-glucose pyrophosphorylase isoforms essential for starch synthesis in the leaf and seed endosperm of rice (Oryza sativa L.).

  Lee S.K., Hwang S.K., Han M., Eom J.S., Kang H.G., Han Y., Choi S.B., Cho M.H., Bhoo S.H., An G., Hahn T.R., Okita T.W., Jeon J.S.

  ADP-glucose pyrophosphorylase (AGP) catalyzes the first committed step of starch biosynthesis in higher plants. To identify AGP isoforms essential for this biosynthetic process in sink and source tissues of rice plants, we analyzed the rice AGP gene family which consists of two genes, OsAGPS1 and ... >> More

  ADP-glucose pyrophosphorylase (AGP) catalyzes the first committed step of starch biosynthesis in higher plants. To identify AGP isoforms essential for this biosynthetic process in sink and source tissues of rice plants, we analyzed the rice AGP gene family which consists of two genes, OsAGPS1 and OsAGPS2, encoding small subunits (SSU) and four genes, OsAGPL1, OsAGPL2, OsAGPL3 and OsAGPL4, encoding large subunits (LSU) of this enzyme heterotetrameric complex. Subcellular localization studies using green fluorescent protein (GFP) fusion constructs indicate that OsAGPS2a, the product of the leaf-preferential transcript of OsAGPS2, and OsAGPS1, OsAGPL1, OsAGPL3, and OsAGPL4 are plastid-targeted isoforms. In contrast, two isoforms, SSU OsAGPS2b which is a product of a seed-specific transcript of OsAGPS2, and LSU OsAGPL2, are localized in the cytosol. Analysis of osagps2 and osagpl2 mutants revealed that a lesion of one of the two cytosolic isoforms, OsAGPL2 and OsAGPS2b, causes a shrunken endosperm due to a remarkable reduction in starch synthesis. In leaves, however, only the osagps2 mutant appears to severely reduce the transitory starch content. Interestingly, the osagps2 mutant was indistinguishable from wild type during vegetative plant growth. Western blot analysis of the osagp mutants and wild type plants demonstrated that OsAGPS2a is an SSU isoform mainly present in leaves, and that OsAGPS2b and OsAGPL2 are the major SSU and LSU isoforms, respectively, in the endosperm. Finally, we propose a spatiotemporal complex model of OsAGP SSU and LSU isoforms in leaves and in developing endosperm of rice plants. << Less

  Plant Mol. Biol. 65:531-546(2007) [PubMed] [EuropePMC]

• Conserved residues of the Pro103-Arg115 loop are involved in triggering the allosteric response of the Escherichia coli ADP-glucose pyrophosphorylase.

  Hill B.L., Wong J., May B.M., Huerta F.B., Manley T.E., Sullivan P.R., Olsen K.W., Ballicora M.A.

  The synthesis of glycogen in bacteria and starch in plants is allosterically controlled by the production of ADP-glucose by ADP-glucose pyrophosphorylase. Using computational studies, site-directed mutagenesis, and kinetic characterization, we found a critical region for transmitting the allosteri ... >> More

  The synthesis of glycogen in bacteria and starch in plants is allosterically controlled by the production of ADP-glucose by ADP-glucose pyrophosphorylase. Using computational studies, site-directed mutagenesis, and kinetic characterization, we found a critical region for transmitting the allosteric signal in the Escherichia coli ADP-glucose pyrophosphorylase. Molecular dynamics simulations and structural comparisons with other ADP-glucose pyrophosphorylases provided information to hypothesize that a Pro103-Arg115 loop is part of an activation path. It had strongly correlated movements with regions of the enzyme associated with regulation and ATP binding, and a network analysis showed that the optimal network pathways linking ATP and the activator binding Lys39 mainly involved residues of this loop. This hypothesis was biochemically tested by mutagenesis. We found that several alanine mutants of the Pro103-Arg115 loop had altered activation profiles for fructose-1,6-bisphosphate. Mutants P103A, Q106A, R107A, W113A, Y114A, and R115A had the most altered kinetic profiles, primarily characterized by a lack of response to fructose-1,6-bisphosphate. This loop is a distinct insertional element present only in allosterically regulated sugar nucleotide pyrophosphorylases that could have been acquired to build a triggering mechanism to link proto-allosteric and catalytic sites. << Less

  Protein Sci 24:714-728(2015) [PubMed] [EuropePMC]

• Biosynthesis of bacterial glycogen. Mutagenesis of a catalytic site residue of ADP-glucose pyrophosphorylase from Escherichia coli.

  Hill M.A., Kaufmann K., Otero J., Preiss J.

  Site-directed mutagenesis was used to explore the role of Lys-195 in ADP-glucose pyrophosphorylase from Escherichia coli. This residue, which is conserved in every bacterial and plant source sequenced to date, was originally identified as a potential catalytic site residue by covalent modification ... >> More

  Site-directed mutagenesis was used to explore the role of Lys-195 in ADP-glucose pyrophosphorylase from Escherichia coli. This residue, which is conserved in every bacterial and plant source sequenced to date, was originally identified as a potential catalytic site residue by covalent modification studies. Mutation of Lys-195 to glutamine produces an enzyme whose Km for glucose 1-phosphate is 600-fold greater than that measured for the wild-type enzyme. The effect on glucose 1-phosphate is very specific since kinetic constants measured for ATP, Mg2+, and the allosteric activator, fructose 1,6-bisphosphate, are unchanged relative to those measured for the wild-type enzyme. Furthermore, the catalytic rate constant, Kcat, for the glutamine mutant is similar to that of the wild-type enzyme. Taken together, the results suggest a role for Lys-195 in binding of glucose 1-phosphate and exclude its role as a participant in the rate-determining step(s) in the catalytic reaction mechanism. To further study the effect of charge, shape, size, and hydrophobicity of the amino acid residue at position 195, a series of mutants were prepared including arginine, histidine, isoleucine, and glutamic acid. In every case, the kinetic constants measured for ATP, Mg2+, and fructose 1,6-bisphosphate were similar to wild-type constants, reinforcing the notion that this residue is responsible for a highly localized effect at the glucose 1-phosphate-binding site and also suggesting that the protein can accommodate a wide range of substitutions at this position without losing its global folding properties. Thermal stability measurements corroborate this finding. The mutations did, however, produce a range of glucose 1-phosphate Km values from 100-to 10,000-fold greater than wild-type, which indicate that both size and charge properties of lysine are essential for proper binding of glucose 1-phosphate at the catalytic site. AMP binding was also affected by the nature of the mutation at position 195. A model for glucose 1-phosphate, ATP, and AMP binding is presented. << Less

  J. Biol. Chem. 266:12455-12460(1991) [PubMed] [EuropePMC]