An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase

Yep, Alejandra; Bejar, Clarisa M.; Ballícora, Miguel A.; Dubay, Jennifer R.; Iglesias, Alberto A.; Preiss, Jack

doi:10.1016/j.ab.2003.09.024

Cited by 21 publications

(29 citation statements)

References 30 publications

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“…All other reagents were purchased at the highest quality available. Escherichia coli glycogen synthase for the ADP-Glc synthesis assay was expressed and purified as described (18).…”

Section: ␣-D-[u-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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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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“…All other reagents were purchased at the highest quality available. Escherichia coli glycogen synthase for the ADP-Glc synthesis assay was expressed and purified as described (18).…”

Section: ␣-D-[u-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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“…The synthesis of ADP-[ 14 C]Glc from [ 14 C]Glc1P and ATP was measured by the method of Yep et al (56). The standard aqueous reaction mixture contained 50 mM HEPES buffer (pH 8.0), 7 mM MgCl 2 , 0.5 mM […”

Section: Materials ␣-D-[u-mentioning

confidence: 99%

Identification of Regions Critically Affecting Kinetics and Allosteric Regulation of theEscherichia coliADP-Glucose Pyrophosphorylase by Modeling and Pentapeptide-Scanning Mutagenesis

et al. 2007

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ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of the Escherichia coli ADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene. We obtained 140 modified plasmids with single insertions of which 21 were in the coding region of the enzyme. Fourteen of them generated insertions of five amino acids, whereas the other seven created a stop codon and produced truncations. Correlation of ADP-Glc PPase activity to these modifications validated the enzyme model. Six of the insertions and one truncation produced enzymes with sufficient activity for the E. coli cells to synthesize glycogen and stain in the presence of iodine vapor. These were in regions away from the substrate site, whereas the mutants that did not stain had alterations in critical areas of the protein. The enzyme with a pentapeptide insertion between Leu 102 and Pro 103 was catalytically competent but insensitive to activation. We postulate this region as critical for the allosteric regulation of the enzyme, participating in the communication between the catalytic and regulatory domains.

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“…The activity of many higher plant ADP-Glc PPases is allosterically activated by 3-phosphoglycerate (3-PGA) and inhibited by inorganic phosphate (Preiss, 1973;Sivak and Preiss, 1998). Most bacterial enzymes are homotetramers composed of four identical subunits (a 4 ; Haugen et al, 1976;, while higher plant ADP-Glc PPases are heterotetramers composed of two closely related types of subunits (S 2 L 2 ; Morell et al, 1987;Okita et al, 1990;Preiss et al, 1991;Smith-White and Preiss, 1992;Ballicora et al, 2004). In higher plants, a number of studies suggest that the regulatory properties of ADP-Glc PPase are a product of synergistic interactions between the two types of subunits (Ballicora et al, 1998; Slattery et al, 2000;Cross et al, 2004;Hwang et al, 2005;Ventriglia et al, 2007).…”

mentioning

confidence: 99%

“…ADP-Glc is the donor for glycogen synthesis in bacteria and starch synthesis in plants (Sivak and Preiss, 1998; Slattery et al, 2000). ADP-Glc PPase is an allosteric enzyme regulated by intermediates of the major pathway of carbon assimilation in the organism (Ballicora et al, , 2004. The activity of many higher plant ADP-Glc PPases is allosterically activated by 3-phosphoglycerate (3-PGA) and inhibited by inorganic phosphate (Preiss, 1973;Sivak and Preiss, 1998).…”

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confidence: 99%

Two Arabidopsis ADP-Glucose Pyrophosphorylase Large Subunits (APL1 and APL2) Are Catalytic

et al. 2008

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ADP-glucose (Glc) pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (a 2 b 2 ) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants result from the synergy of both types of subunits. In Arabidopsis (Arabidopsis thaliana), two genes encode small subunits (APS1 and APS2) and four large subunits (APL1-APL4). Here, we show that in Arabidopsis, APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a noncatalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-phosphoglycerate). Mutation of the Glc-1-P binding site of Arabidopsis and potato (Solanum tuberosum) isoforms confirmed these observations. To determine the relevance of these activities in planta, a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacks ADP-Glc PPase activity, APS1 mRNA, and APS1 protein, and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild-type phenotype, indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glc synthesis in planta.ADP-Glc pyrophosphorylase (ADP-Glc PPase; EC 2.7.7.27) is a heterotetrameric enzyme that catalyzes the synthesis of ADP-Glc and inorganic pyrophosphate from Glc-1-P and ATP (Espada, 1962). ADP-Glc is the donor for glycogen synthesis in bacteria and starch synthesis in plants (Sivak and Preiss, 1998; Slattery et al., 2000). ADP-Glc PPase is an allosteric enzyme regulated by intermediates of the major pathway of carbon assimilation in the organism (Ballicora et al., , 2004. The activity of many higher plant ADP-Glc PPases is allosterically activated by 3-phosphoglycerate (3-PGA) and inhibited by inorganic phosphate (Preiss, 1973;Sivak and Preiss, 1998). Most bacterial enzymes are homotetramers composed of four identical subunits (a 4 ; Haugen et al., 1976;, while higher plant ADP-Glc PPases are heterotetramers composed of two closely related types of subunits (S 2 L 2 ; Morell et al., 1987;Okita et al., 1990;Preiss et al., 1991;Smith-White and Preiss, 1992;Ballicora et al., 2004). In higher plants, a number of studies suggest that the regulatory properties of ADP-Glc PPase are a product of synergistic interactions between the two types of subunits (Ballicora et al., 1998; Slattery et al., 2000;Cross et al., 2004;Hwang et al., 2005;Ventriglia et al., 2007). Besides, recent evidence suggests that some L subunits may have catalytic activity or influence the net catalysis of the enzyme (Burger et al., 2003). It has been reported that potato (Solanum tuberosum) tuber L subunit can be turned catalytically active by mutagenesis (Ballicora et al., 2005;Hwang et al., 2008) and that mutation of potato tuber L subunit (PLS) amino acid P44 (number...

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An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase

Cited by 21 publications

References 30 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

Identification of Regions Critically Affecting Kinetics and Allosteric Regulation of theEscherichia coliADP-Glucose Pyrophosphorylase by Modeling and Pentapeptide-Scanning Mutagenesis

Two Arabidopsis ADP-Glucose Pyrophosphorylase Large Subunits (APL1 and APL2) Are Catalytic

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