title: The rice endosperm ADP-glucose pyrophosphorylase large subunit is essential for optimal catalysis and allosteric regulation of the heterotetrameric enzyme.
abstract: 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.
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USAThese authors contributed equally to this work.
authors.forename: Aytug
authors.initials: A
authors.lastname: Tuncel
authors.affiliation: Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 JapanThese authors contributed equally to this work.
authors.forename: Joe
authors.initials: J
authors.lastname: Kawaguchi
authors.affiliation: Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan.
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Ihara
authors.affiliation: Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Matsusaka
authors.affiliation: Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan.
authors.forename: Aiko
authors.initials: A
authors.lastname: Nishi
authors.affiliation: Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan.
authors.forename: Tetsuhiro
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Department of Genetic Resources Technology, Kyushu University, Fukuoka, 812-8581 Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Kuhara
authors.affiliation: Kazusa DNA Research Institute, Department of Plant Genome Research, Kisarazu, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Hirakawa
authors.affiliation: Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, 010-0195 Japan.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Nakamura
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA.
authors.forename: Bilal
authors.initials: B
authors.lastname: Cakir
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USAFaculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Nagamine
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA okita@wsu.edu.
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Okita
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA.
authors.forename: Seon-Kap
authors.initials: SK
authors.lastname: Hwang
authors.affiliation: Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Satoh
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D002384:Catalysis; D018389:Codon, Nonsense; D056625:Endosperm / Q000201:enzymology / Q000235:genetics; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007527:Isoenzymes; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D010641:Phenotype; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D058105:Polymerization; D011994:Recombinant Proteins; D012639:Seeds / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D013213:Starch / Q000378:metabolism*
chemical_list: D018389:Codon, Nonsense; D007527:Isoenzymes; D010940:Plant Proteins; D011994:Recombinant Proteins; D013213:Starch; D051599:Glucose-1-Phosphate Adenylyltransferase
keywords: ADP-glucose pyrophosphorylase; Rice endosperm; Starch metabolism