Unraveling the Activation Mechanism of the Potato Tuber ADP-glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase regulates the synthesis of glycogen in bacteria and of starch in plants. The enzyme from plants is mainly activated by 3-phosphoglycerate and is a heterotetramer comprising two small and two large subunits. Here, we found that two highly conserved residues are critical for triggering the activation of the potato tuber ADP-glucose pyrophosphorylase, as shown by site-directed mutagenesis. Mutations in the small subunit, which bears the catalytic function in this potato tuber form, had a more dramatic effect on disrupting the allosteric activation than those introduced in the large subunit, which is mainly modulatory. Our results strongly agree with a model where the modified residues are located in loops responsible for triggering the allosteric activation signal for this enzyme, and the sensitivity to this activation correlates with the dynamics of these loops. In addition, previous biochemical data indicates that the triggering mechanism is widespread in the enzyme family, even though the activator and the quaternary structure are not conserved.Fil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;Fil: Kuhn, Misty L.. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Falaschetti, Christine A.. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Solamen, Ligin. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Olsen, Kenneth W.. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Ballicora, Miguel A.. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina

On the Roles of Wheat Endosperm ADP-Glucose Pyrophosphorylase Subunits

The ADP-glucose pyrophosphorylase from wheat endosperm controls starch synthesis in seeds and has unique regulatory properties compared to others from this family. It comprises two types of subunits, but despite its importance little is known about their roles. Here, we synthesized de novo the wheat endosperm ADP-glucose pyrophosphorylase small (S) and large (L) subunit genes, heterologously expressed them in Escherichia coli, and kinetically characterized the recombinant proteins. To understand their distinct roles, we co-expressed them with well characterized subunits from the potato tuber enzyme to obtain hybrids with one S subunit from one source and an L subunit from the other. After kinetic analyses of these hybrids, we concluded that the unusual insensitivity to activation of the wheat endosperm enzyme is caused by a pre-activation of the L subunit. In addition, the heat stability and sensitivity to phosphate are given by the S subunit

Consumption of fish oil providing amounts of eicosapentaenoic acid and docosahexaenoic acid that can be obtained from the diet reduces blood pressure in adults with systolic hypertension: a retrospective analysis

Background: Although a large number of randomized controlled trials (RCTs) have examined the impact of the n-3 (ω-3) fatty acids EPA (20:5n-3) and DHA (22:6n-3) on blood pressure and vascular function, the majority have used doses of EPA+DHA of > 3 g per d,which are unlikely to be achieved by diet manipulation. Objective: The objective was to examine, using a retrospective analysis from a multi-center RCT, the impact of recommended, dietary achievable EPA+DHA intakes on systolic and diastolic blood pressure and microvascular function in UK adults. Design: Healthy men and women (n = 312) completed a double-blind, placebo-controlled RCT consuming control oil, or fish oil providing 0.7 g or 1.8 g EPA+DHA per d in random order each for 8 wk. Fasting blood pressure and microvascular function (using Laser Doppler Iontophoresis) were assessed and plasma collected for the quantification of markers of vascular function. Participants were retrospectively genotyped for the eNOS rs1799983 variant. Results: No impact of n-3 fatty acid treatment or any treatment * eNOS genotype interactions were evident in the group as a whole for any of the clinical or biochemical outcomes. Assessment of response according to hypertension status at baseline indicated a significant (P=0.046) fish oil-induced reduction (mean 5 mmHg) in systolic blood pressure specifically in those with isolated systolic hypertension (n=31). No dose response was observed. Conclusions: These findings indicate that, in those with isolated systolic hypertension, daily doses of EPA+DHA as low as 0.7 g bring about clinically meaningful blood pressure reductions which, at a population level, would be associated with lower cardiovascular disease risk. Confirmation of findings in an RCT where participants are prospectively recruited on the basis of blood pressure status is required to draw definite conclusions

Kinetic parameters obtained for Pi with the potato tuber ADP-Glc PPase and its mutants.

<p>Reactions were performed using <i>Assay B</i> in absence or presence of 5 mM 3-PGA, as stated under “Materials and Methods”.</p

Saturation curves of 3-PGA for the wild type potato tuber ADP-Glc PPase (▪) and mutants <i>Stu</i>S_Q75A/<i>Stu</i>L (•), <i>Stu</i>S/<i>Stu</i>L_Q86A (○), <i>Stu</i>S_W116A/<i>Stu</i>L (▴), <i>Stu</i>S/<i>Stu</i>L_W128A (▵), and <i>Stu</i>S_W116A/<i>Stu</i>L_W128A (▾).

<p>Reactions were performed using <i>Assay B</i>, as stated under “Materials and Methods”.</p

Changes in the molecular dynamics simulations due to mutations in the subunits.

<p>The structures are colored according to the difference RMSF values. Colors used are purple, more than 3 standard deviations negative; dark blue, 2 to 3 standard deviations negative; light blue, 1 to 2 standard deviations negative; yellow, +/− one standard deviation; pink, 1 to 2 standard deviations positive; orange, 2 to 3 standard deviations positive; and red, more than 3 standard deviations positive. A. <i>Stu</i>S_Q75A. B. <i>Stu</i>L_Q86A. C. <i>Stu</i>S_W116A. D. <i>Stu</i>L_W128A. The position of the residue mutated to Ala is shown in red, while the non-mutated position is shown in green. The values of the standard deviations were <i>Stu</i>S_Q75A, 0.38; <i>Stu</i>L_Q86A, 0.40; <i>Stu</i>S_W116A, 0.40; and <i>Stu</i>L_W128A, 0.43. Because of the different flexibilities, the Trp-containing loop that was mutated is in different colors. It is shown in red in panel D (arrow).</p

Kinetic parameters obtained for 3-PGA with the<i>Anabaena</i> and potato tuber ADP-Glc PPases and their mutants.

<p>Reactions for the <i>Anabaena</i> and potato tuber enzymes were performed using <i>Assay A</i> and <i>B</i>, respectively, as stated under “Materials and Methods”.</p