ADP-Glucose Pyrophosphorylase from Potato Tubers. Site-Directed Mutagenesis Studies of the Regulatory Sites1

Ballícora, Miguel A.; Fu, Yingbin; Nesbitt, Natasha M.; Preiss, Jack

doi:10.1104/pp.118.1.265

Cited by 66 publications

(72 citation statements)

References 29 publications

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“…4). This activity was lower but on the same order of magnitude as those reported for the corresponding potato tuber recombinant enzymes (30 and 32 units/mg) (1). Similarly, the recombinant heterotetramer maize endosperm was 26.7 units/mg (40).…”

Section: Resultsmentioning

confidence: 69%

“…It is not clear if there is a set of universal roles for the L subunit in plants. The L subunit from potato tuber (StuL) is catalytically deficient and plays more of a regulatory role by modifying the apparent affinity of the S subunit toward allosteric regulators (1,4). On the other hand, the Arabidopsis APL1 and APL2 isoforms have both catalytic and regulatory functions, whereas the APL3 and APL4 isoforms behave like StuL (2,5).…”

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confidence: 99%

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Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles

Kuhn¹,

Falaschetti²,

Ballícora

2009

Journal of Biological Chemistry

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ADP-glucose pyrophosphorylase controls starch synthesis in plants and is an interesting case to study the evolution and differentiation of roles in heteromeric enzymes. It includes two homologous subunits, small (S) and large (L), that originated from a common photosynthetic eukaryotic ancestor. In present day organisms, these subunits became complementary after loss of certain roles in a process described as subfunctionalization. For instance, the potato tuber enzyme has a noncatalytic L subunit that complements an S subunit with suboptimal allosteric properties. To understand the evolution of catalysis and regulation in this family, we artificially synthesized both subunit genes from the unicellular alga Ostreococcus tauri. This is among the most ancient species in the green lineage that diverged from the ancestor of all green plants and algae. After heterologous gene expression, we purified and characterized the proteins. The O. tauri enzyme was not redox-regulated, suggesting that redox regulation of ADP-glucose pyrophosphorylases appeared later in evolution. The S subunit had a typical low apparent affinity for the activator 3-phosphoglycerate, but it was atypically defective in the catalytic efficiency (V max /K m ) for the substrate Glc-1-P. The L subunit needed the S subunit for soluble expression. In the presence of a mutated S subunit (to avoid interference), the L subunit had a high apparent affinity for 3-phosphoglycerate and substrates suggesting a leading role in catalysis. Therefore, the subfunctionalization of the O. tauri enzyme was different from previously described cases. To the best of our knowledge, this is the first biochemical description of a system with alternative subfunctionalization paths.Starch synthesis in photosynthetic eukaryotes such as higher plants and unicellular algae is controlled by a heterotetrameric ADP-glucose pyrophosphorylase (ADP-Glc PPase, 4 EC 2.7.7.27). This enzyme catalyzes the reaction of ATP and Glc-1-P to form ADP-glucose (ADP-Glc) and PP i , and it is primarily activated by 3-phosphoglycerate (3-PGA) and inhibited by P i . In photosynthetic eukaryotes the enzyme includes two distinct S and L homologous subunits (S 2 L 2 or ␣ 2 ␤ 2 ), but in photosynthetic bacteria the enzyme is a homotetramer (␣ 4 ). There has been debate regarding the role of the different subunits of ADP-Glc PPase. The S subunit homotetramer from potato (Solanum tuberosum) tuber (StuS), Arabidopsis thaliana (APS1), and barley (Hordeum vulgare) endosperm have a catalytic function with defective regulatory properties (1-3). It is not clear if there is a set of universal roles for the L subunit in plants. The L subunit from potato tuber (StuL) is catalytically deficient and plays more of a regulatory role by modifying the apparent affinity of the S subunit toward allosteric regulators (1, 4). On the other hand, the Arabidopsis APL1 and APL2 isoforms have both catalytic and regulatory functions, whereas the APL3 and APL4 isoforms behave like StuL (2, 5). The maize (Zea mays) endosperm L subunit...

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Section: Resultsmentioning

confidence: 69%

mentioning

confidence: 99%

Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles

Kuhn¹,

Falaschetti²,

Ballícora

2009

Journal of Biological Chemistry

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“…27) Although the expression of these mutant enzymes in transgenic Arabidopsis exhibited higher starch content than plants expressing ALS-WT at the end of light 10.0 (39) 5.4 (21) 1.9 (7) 3.5 (11) 3.5 (13) All enzyme activities were determined from ADP-glucose synthesis assay data of three independent experiments, and the SE were within 15% in all cases. Specific activities (U/mg) of the enzymes are presented.…”

Section: Activation Of Mutated Agpases By Various Sugar Metabolitesmentioning

confidence: 99%

“…[9][10][11][12][13] The specific phosphate ester molecule is evolutionarily adapted to controlling carbon flux in the major carbon metabolic pathway. The results obtained here show the possibility of extending the specificity of the allosteric effectors of plant AGPase activity, and in turn of increasing the net catalytic activity of AGPase for enhanced starch production.…”

Section: Activation Of Mutated Agpases By Various Sugar Metabolitesmentioning

confidence: 99%

“…9,10) On the other hand, the activator and the inhibitor of higher plant AGPases are 3-phosphoglycerate (3-PGA) and orthophosphate (Pi) respectively. [11][12][13] In contrast to bacterial AGPase, a homotetrameric protein, the higher plant AGPase is composed of two small subunits (SSs) and two large subunits (LSs), which form a heterotetramer. [14][15][16] LS and SS share considerable sequence homology but have distinct roles in catalysis and allosteric regulation, as best exemplified by the potato tuber AGPase.…”

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Modulation of Allosteric Regulation by E38K and G101N Mutations in the Potato Tuber ADP-glucose Pyrophosphorylase

Wakuta

Shibata

Yoshizaki

et al. 2013

Bioscience, Biotechnology, and Biochemistry

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Starch Biosynthesis in Plants

Preiss

2008

Wiley Encyclopedia of Chemical Biology

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Starch is a major storage compound in plants that is present both in leaves and in storage tissues. Biochemical and molecular biological data show that ADP‐glucose is the glucosyl donor for plant starch synthesis, and its synthesis is catalyzed by ADP‐glucose pyrophosphorylase. Subsequently, starch synthases catalyze the transfer of the glucosyl residue from ADP‐glucose to the oligosaccharide chains of the starch components amylose and amylopectin to form new α‐1,4‐glucosidic residues. After elongation of these α‐1,4‐glucosidic chains, the branching enzyme catalyzes a cleavage of the elongated chain and transfers the cleaved portion of the oligosaccharide chain to either another region in the amylopectin molecule or to a new amylopectin and forms a new α‐1,6‐glucosidic linkage. Amylose synthesis is catalyzed by the granule‐bound starch synthase. Regulation of starch synthesis occurs at the ADP‐glucose pyrophosphorylase step. The enzyme from higher plants, green algae, and cyanobacteria is activated allosterically by 3‐phosphoglycerate and inhibited by inorganic phosphate. Isolation of mutants and control analyses indicate that the allosteric activation and inhibition are of physiological and functional importance in the regulation of starch synthesis. Furthermore, evidence indicates that ADP‐glucose pyrophosphorylases can also be regulated by a redox mechanism. The current knowledge of the enzyme structures and critical amino acids necessary for substrate binding, allosteric effector binding, regulation, and catalysis for the ADP‐glucose pyrophosphorylase is reviewed.

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ADP-Glucose Pyrophosphorylase from Potato Tubers. Site-Directed Mutagenesis Studies of the Regulatory Sites1

Cited by 66 publications

References 29 publications

Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles

Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles

Modulation of Allosteric Regulation by E38K and G101N Mutations in the Potato Tuber ADP-glucose Pyrophosphorylase

Starch Biosynthesis in Plants

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