Phylogenetic Analysis of ADP-Glucose Pyrophosphorylase Subunits Reveals a Role of Subunit Interfaces in the Allosteric Properties of the Enzyme

Georgelis, Nikolaos; Shaw, Janine R.; Hannah, L. Curtis

doi:10.1104/pp.109.138933

Cited by 48 publications

(30 citation statements)

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“…The potato and A. tumefaciens AGPases respond to different allosteric effectors and these sequence differences may be required to accommodate the different ligands. Full alignments of these sequences have been published and analyzed previously (Georgelis et al, 2007(Georgelis et al, , 2008(Georgelis et al, , 2009). …”

Section: Rationale For Mutagenesismentioning

confidence: 99%

Probing Allosteric Binding Sites of the Maize Endosperm ADP-Glucose Pyrophosphorylase

et al. 2009

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Maize (Zea mays) endosperm ADP-glucose pyrophosphorylase (AGPase) is a highly regulated enzyme that catalyzes the rate-limiting step in starch biosynthesis. Although the structure of the heterotetrameric maize endosperm AGPase remains unsolved, structures of a nonnative, low-activity form of the potato tuber (Solanum tuberosum) AGPase (small subunit homotetramer) reported previously by others revealed that several sulfate ions bind to each enzyme. These sites are also believed to interact with allosteric regulators such as inorganic phosphate and 3-phosphoglycerate (3-PGA). Several arginine (Arg) side chains contact the bound sulfate ions in the potato structure and likely play important roles in allosteric effector binding. Alanine-scanning mutagenesis was applied to the corresponding Arg residues in both the small and large subunits of maize endosperm AGPase to determine their roles in allosteric regulation and thermal stability. Steady-state kinetic and regulatory parameters were measured for each mutant. All of the Arg mutants examined—in both the small and large subunits—bound 3-PGA more weakly than the wild type (A 50 increased by 3.5- to 20-fold). By contrast, the binding of two other maize AGPase allosteric activators (fructose-6-phosphate and glucose-6-phosphate) did not always mimic the changes observed for 3-PGA. In fact, compared to 3-PGA, fructose-6-phosphate is a more efficient activator in two of the Arg mutants. Phosphate binding was also affected by Arg substitutions. The combined data support a model for the binding interactions associated with 3-PGA in which allosteric activators and inorganic phosphate compete directly.

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Section: Rationale For Mutagenesismentioning

confidence: 99%

Probing Allosteric Binding Sites of the Maize Endosperm ADP-Glucose Pyrophosphorylase

et al. 2009

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“…In particular, the S163F mutation enhances the thermodynamic stability of AGPase. Unfortunately, it also decreases the K i for phosphate inhibition and increases the K a for 3-PGA and the K m for ATP [33]. These properties make the S163F mutation a less-desirable catalyst for agronomic applications.…”

Section: Discussionmentioning

confidence: 92%

“…The same sites were also highlighted as potentially important from evolutionary considerations (Fig. 2) [33]. Changing S444 to Ala yielded more darkly-staining colonies when expressed in E. coli, a 49% increase in k cat at 37°C, and a 50% reduction in the ATP K m .…”

Section: Discussionmentioning

confidence: 97%

“…2). Phylogenetic analysis of plant large subunits illustrated that this amino acid position shows what is termed Type II selection or family specific selection [33]. One amino acid at a particular position is conserved in the isoform expressed in one family; whereas, a different amino acid at this position is conserved in the isoform expressed in another family.…”

Section: Discussionmentioning

confidence: 98%

“…These criteria yielded nine residues in the structure of 1YP2. The corresponding amino acids in both the small and large subunit of maize endosperm AGPase were determined by using the previously-published alignment [33] and the residues are shown in Table 1.…”

Section: Plasmid Preparationmentioning

confidence: 99%

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Enhancing the heat stability and kinetic parameters of the maize endosperm ADP-glucose pyrophosphorylase using iterative saturation mutagenesis

Boehlein

Shaw

Stewart

et al. 2015

Archives of Biochemistry and Biophysics

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A brittle‐2 transgene increases maize yield by acting in maternal tissues to increase seed number

et al. 2017

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The enzyme ADP-glucose pyrophosphorylase is essential for starch biosynthesis and is highly regulated. Here, mutations that increased heat stability and interactions with allosteric effectors were incorporated into the small subunit of the isoform known to be expressed at high levels in the maize endosperm. The resulting variants were transformed into maize with expression targeted to the endosperm. Transgenes harboring the changes increased yield some 35%; however, yield enhancement occurred via an increase in seed number rather than by increased seed weight. Interestingly, seed number increase is controlled by the genotype of the plant rather than the genotype of the seed as seeds increase in number whether or not they contain the transgene as long as the maternal parent has the transgene.The transgene is however expressed in the endosperm, and the altered allosteric and stability properties initially seen in Escherichia coli expression experiments are also seen with the endosperm-expressed gene. The extent of seed number increase is positively correlated with the average daily high temperature during the first 4 days postpollination. While these results were unexpected, they echo the phenotypic changes caused by the insertion of an altered large subunit of this enzyme reported previously (Plant Cell, 24, 2012, 2352. These results call into question some of the reported fundamental differences separating starch synthesis in the endosperm vis-a-vis other plant tissues.abiotic plant stress, ADP-glucose pyrophosphorylase, maize yield, starch synthesis | INTRODUCTIONStarch is the major component of cereal seeds and hence a critical constituent of cereal seed yields. The importance of starch, coupled with the early availability of seminal mutants in important biosynthetic steps, has allowed for a fairly robust understanding of its biochemical pathway (reviewed in Smith, Denyer, & Martin, 1997;Ballicora, Iglesias, & Preiss, 2003;Hannah, 2007;Hannah & Greene, 2008;Hannah & James, 2008;Preiss, 2009;Keeling & Myers, 2010).A series of studies have pointed to the enzyme, ADP-glucose pyrophosphorylase (AGPase) as a key step in this important pathway.AGPase is composed of two identical small and two identical large subunits and synthesizes ADP-glucose from glucose-1-PO 4 and ATP.The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plant physiol.org) is L. Curtis Hannah (lchannah@ufl.edu). inbred B73 and were analyzed in several growing seasons. Complementation of the bt2 mutation was evidenced by a 15 plump: 1 shrunken seed ratio in progeny derived by self-pollination of the F1 generation. The presence of the transgene was monitored by painting a small portion of one leaf with Basta 7 to 10 days postpollination and scoring for resistance 7 days later. Selected transgenic events were also monitored for cosegregation of mutant seed with herbicide sensitivity. All materials for analysis were ...

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Phylogenetic Analysis of ADP-Glucose Pyrophosphorylase Subunits Reveals a Role of Subunit Interfaces in the Allosteric Properties of the Enzyme

Cited by 48 publications

References 60 publications

Probing Allosteric Binding Sites of the Maize Endosperm ADP-Glucose Pyrophosphorylase

Probing Allosteric Binding Sites of the Maize Endosperm ADP-Glucose Pyrophosphorylase

Enhancing the heat stability and kinetic parameters of the maize endosperm ADP-glucose pyrophosphorylase using iterative saturation mutagenesis

A brittle‐2 transgene increases maize yield by acting in maternal tissues to increase seed number

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